Naphthalene-based inhibitors of anti-apoptotic proteins

ABSTRACT

Methods of using apogossypol and its derivatives for treating inflammation is disclosed. Also, there is described a group of compounds having structure A, or a pharmaceutically acceptable salt, hydrate, N-oxide, or solvate thereof are provided: 
                         
wherein each R is independently H, C(O)X, C(O)NHX, NH(CO)X, SO 2 NHX, or NHSO 2 X, wherein X is hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, alkylaryl, substituted alkylaryl, heterocycle, or substituted heterocycle. Compounds of group A may be used for treating various diseases or disorders, such as cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 12/253,918 filed on Oct. 17, 2008, and also claims the benefitof priority under 35 U.S.C. §119(e) to U.S. Provisional Application No.61/254,172 filed on Oct. 22, 2009, and to U.S. Provisional ApplicationNo. 61/169,686, filed on Apr. 15, 2009, the disclosure of each of whichis hereby incorporated by reference in their entirety for all purposes.

GRANT INFORMATION

This invention was made in part with government support under NIH (GrantU01 A1061139 and Grant CA113318), and CSRA (Grant No. 08-02). The UnitedStates Government has certain rights in this invention.

FIELD OF THE DISCLOSURE

The disclosure relates generally to a class of compounds derived fromnaphthalene, such as apogossypol and derivatives thereof, for treating avariety of disorders, diseases and pathologic conditions, and morespecifically, for treating cancer, autoimmune diseases, and/orinflammation.

BACKGROUND OF THE DISCLOSURE

The apoptotic cascade in cells is known to lead to cell death. Whenanti-apoptotic proteins, such as BCL-2 family proteins, are overproducedby the cells, uncontrollable cell growth may ensue, potentially leadingto the development of various serious diseases, disorders, andpathologies, particularly cancer. Programmed cell-death (apoptosis)plays critical roles in the maintenance of normal tissue homeostasis,ensuring a proper balance of cell production and cell loss. Defects inthe regulation of programmed cell death promote tumorgenesis, and alsocontribute significantly to chemoresistance. Bcl-2 (B-celllymphoma/leukemia-2) family proteins are central regulators ofapoptosis. In humans, six anti-apoptotic members of the Bcl-2 familyhave been identified and characterized thus far, including Bcl-2,Bcl-X_(L), Mcl-1, Bfl-1, Bcl-W and Bcl-B. Over-expression ofanti-apoptotic Bcl-2 family proteins occurs in many human cancers andleukemias, and therefore these proteins are very attractive targets forthe development of novel anticancer agents. Members of the Bcl-2 familyproteins also include pro-apoptotic effectors such as Bak, Bax, Bad, Bimand Bid. Anti-apoptotic and pro-apoptotic Bcl-2 family proteins dimerizeand negate each other's functions. Structural studies have elucidated ahydrophobic crevice on the surface of anti-apoptotic Bcl-2 familyproteins that binds the BH3 dimerization domain of pro-apoptotic familymembers. Thus, molecules that mimic the BH3 domain of pro-apoptoticproteins induce apoptosis and/or abrogate the ability of anti-apoptoticBcl-2 proteins to inhibit cancer cell death.

Apoptosis plays a role in tissue homeostatic, for the physiologicalremoval of unwanted cells during development and in host defensemechanism. The BCL-2 family of proteins are believed to be involved inregulating of apoptosis. Specifically, members of the BCL-2 gene familycan act to inhibit programmed cell death (e.g., BCL-2, BCL-X_(L), ced-9)or promote cell death (e.g., Bax, Bak, BCL-X_(S)). Pro-survival membersof this family, such as BCL-X_(L), contain, on the surface, ahydrophobic groove in which is believed to allow binding of the BH3domain of the pro-apoptotic counterpart. This binding is believed toplay role in apoptosis regulation, in fact pro- and anti-survivalproteins can reverse each other function through dimerization.

Therefore, a need exists to inhibit anti-apoptotic proteins, such as theBCL-2 family proteins. Various potential BCL-2 antagonists have beenpreviously identified. However, none of these compounds inhibits all sixproteins in the BCL-2 family, i.e., all of the following proteins:BCL-X_(L), BCL-2, BCL-W, BCL-B, BFL-1, and MCL-1. For example, none ofthe previously identified synthetic BCL-2 antagonists was effective atinhibiting the protein BFL-1. Therefore, the efficiency of suchantagonists is not as high as desired. In addition, the existingantagonists are characterized by other drawbacks, such as insufficiencyor safety issues.

Defects in the regulation of programmed cell death may promotetumorgenesis, and also contribute to chemoresistance. Over-expression ofanti-apoptotic BCL-2 family proteins occurs in many human cancers andleukemias, and therefore these proteins may be used as targets for thedevelopment of novel anticancer agents. Structural studies haveelucidated a hydrophobic crevice on the surface of anti-apoptotic BCL-2family proteins that binds the BH3 dimerization domain of pro-apoptoticfamily members. Thus, molecules that mimic the BH3 domain ofpro-apoptotic proteins induce apoptosis and/or abrogate the ability ofanti-apoptotic BCL-2 proteins to inhibit cancer cell death.

It has been previously shown that the natural product gossypol shown onFIG. 1A is an inhibitor of BCL-2, BCL-X_(L) and MCL-1, functioning as aBH3 mimic. (−) Gossypol is currently in clinical trails, displayingsingle-agent antitumor activity in patients with advanced malignancies.Given that gossypol has toxicity problems likely due to two reactivealdehyde groups, we prepared apogossypol, a compound that lacks thesealdehydes, but retains activity against anti-apoptotic BCL-2 familyproteins in vitro and in cells has been also evaluated previously.Recently, the efficacy and toxicity in mice of gossypol and apogossypolwere compared. Preclinical in vivo data show that apogossypol has betterefficacy and reduced toxicity compared to gossypol, as well as bettersingle-dose pharmacokinetic characteristics, including, superior bloodconcentrations over time compared to gossypol, due to slower clearance.These observations indicate that apogossypol is a promising leadcompound for cancer therapy.

BCL-2 family members are also believed to be involved in inflammatorydisorders. For example, BCL-2 family members have been shown to playroles in neutrophil apoptosis and inflammatory accumulation. In severalinflammatory diseases, the delay of neutrophil apoptosis is associatedwith reduced levels of the pro-apoptotic BCL-2 family member BAX. It hasbeen also shown that eosinophils isolated from children with acuteasthma had an increased expression of the anti-apoptotic protein BCL-2,which was inversely correlated with expiratory flow rate. BCL-2 familyproteins are also associated with Crohn's disease. BAX expression isattenuated and BCL-X_(L) expression is increased in T cells isolatedfrom the lamina propria from patients with Crohn's disease. This showsthat inflammatory cell survival, by means of prosurvival andanti-apoptotic signaling mechanisms, are involved in the pathogenesis ofinflammatory diseases. Lupus is a complex systemic autoimmune disease,characterized by high levels of anti-DNA and anti-glomerularautoantibodies, activated B and T-cells, and glomerulonephritis.Neutrophils from lupus-susceptible mice display reduced rates ofapoptosis. The decreased apopotosis is associated with the alteredexpression of BCL-2 family proteins contributing to the greateraccumulation of neutrophils in the lupus-susceptible mice. Signalingstudies using several different lupus strains indicate that multiplesignaling pathways are upregulated in lymphocytes and non lymphocytes asdisease evolves, including the activation of BCL-2 and BCL-X_(L). Theseanti-apoptotic molecules are known to prolong the lifespan of all cells,including autoreactive B and T cells.

In view of these drawbacks and deficiencies of existing BCL-2inhibitors, new antagonists of anti-apoptotic proteins, such as BCL-2family proteins, are desired. It is desirable that such new antagonistsbe safer and more effective than the existing compounds.

SUMMARY OF THE DISCLOSURE

The disclosure addresses these needs by providing new antagonists ofanti-apoptotic proteins, including the BCL-2 family of proteins. Thus,in one embodiment the disclosure provides compounds having structure A,or pharmaceutically acceptable salts, hydrates, N-oxides, or solvatesthereof:

wherein each R is independently H, C(O)X, C(O)NHX, NH(CO)X, SO₂NHX, andNHSO₂X, wherein X is an hydroxyl, alkyl, substituted alkyl, aryl,substituted aryl, alkylaryl, substituted alkylaryl, a heterocycle, or asubstituted heterocycle.

According to another embodiment, the disclosure provides a compound thatis a species of the compounds having structure A, the specific compoundhaving the formula I:

According to another embodiment, the disclosure provides a method fortreating cancer or autoimmune diseases, by administering to a subject inneed thereof a therapeutically effective amount of the compounds havingstructure A, including the species I, or pharmaceutically acceptablesalts, hydrates, N-oxides, or solvates thereof.

The disclosure also provides a method for treating inflammation. Inparticular, the disclosure relates to the use of apogossypol for thetreatment of inflammation. Accordingly, a method for treatinginflammation is disclosed. The method includes administering to a mammala compound, in an amount effective to reduce inflammation, the compoundhaving structure B:

wherein each of R⁶, R⁸, R⁹ and R¹⁰ is hydrogen, hydroxyl, —(C₁-C₆)alkyl,—O(C₁-C₆)alkyl, —(C₁-C₆)alkylhalo, —OC(O)(C₁-C₆)alkyl, and halo, andeach R⁷ is hydrogen, —(C₁-C₆)alkyl, —(C₃-C₈)cycloalkyl, —(C₆-C₁₀)aryl,and —(C₁-C₆)alkyl(C₆-C₁₀)aryl, C(O)X, C(O)NHX, NH(CO)X, SO₂NHX, andNHSO₂X, wherein X is hydroxyl, alkyl, substituted alkyl, aryl,substituted aryl, alkylaryl, substituted alkylaryl, heterocycle, orsubstituted heterocycle or a pharmaceutically acceptable salt, hydrate,N-oxide, or solvate thereof. In some embodiments, the compound is usedto treat inflammation is apogossypol, for example, (−) apogossypol thatis substantially free of (+) apogossypol.

Also disclosed is a method of treating inflammation in a subject, byadministering to the subject an anti-inflammatory agent selected fromgossypol, apogossypol, L-apogossypol, derivatives of apogossypol,theaflavin, theaflavin-3′-gallate, theaflavanin, (−)gallocatechin-3-gallate (GCG), (−) epigallocatechin-3-gallate (EGCG),(−) catechin-3-gallate (CG), (−) epicatechin-3-gallate (ECG),derivatives of purpurogallin, and mixtures thereof.

In addition, the disclosure provides a method for inducing apoptosis,modulating caspase activity, or inducing cell death in a mammalsuffering from an inflammatory disease inflammation is disclosed. Themethod comprises contacting the mammal with a compound in the amounteffective to induce apoptosis, modulate caspase activity, or induce celldeath the target cells, the compound to be used having structure B asdescribed herein.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 demonstrates structures of gossypol and apogossypol (A);structure of a compound of the disclosure (B); and molecular dockingstudies (C, D).

FIG. 2 demonstrates, NMR binding studies (A) and inhibiting activity ofsome compounds of the disclosure (B).

FIG. 3 demonstrates effectiveness of compounds of the disclosure onshrinkage of BCL-2 mouse spleen.

FIG. 4 demonstrates FP competitive binding curves of compounds of thedisclosure using BCL-X_(L).

FIGS. 5A and 5B depict toxicity profiles of gossypol vs. apogossypol.

FIGS. 6A-6C depict hematological profiles of mice treated withapogossypol or gossypol.

FIG. 7 depicts reactive blood chemistry profiles of mice treated withapogossypol or gossypol.

FIG. 8 depicts a comparison of apoptosis induction of NHL B-cell lines,including DOHH2, RS11846 and 380, by apogossypol and gossypol.

FIG. 9 depicts a comparison of activity of gossypol and apogossypolagainst cultured murine B-cells from transgenic mice: BCL-2 vs.BCL-2/TRAF2DN.

FIG. 10 depicts a comparison of apogossypol and gossypol induction ofapoptosis of cultured CLL B-cells.

FIGS. 11A and 11B depict apogossypol activity in BCL-2 transgenic mice.

FIG. 12 shows a general synthetic scheme that can be used to synthesizesome compounds of the disclosure.

FIG. 13 shows: (A) Structure of Gossypol (1), Apogossypol (2) andBI79D10 (3). (B) Structure of 5,5′ substituted Apogossypol derivatives.(C) and (D), Molecular docking studies. Stereo views of dockedstructures of (C) compound 2 (Apogossypol) and (D) compound 8r intoBcl-2 (PDB ID:1YSW).

FIG. 14 shows: (A) NMR binding studies. Aliphatic region of the ¹H-NMRspectrum of Bcl-X_(L) (25 μM, black) and Bcl-X_(L) in the presence ofcompound 8m (200 μM, grey), compound 8q (200 μM, blue), and compound 8r(200 μM, red). (B) Fluorescence polarization-based competitive bindingcurves of 8m (solid squares), 8q (solid up triangle), 8r (solid downtriangle) and 2 (Apogossypol) (solid dots) using Bcl-2. (C) Inhibitionof cell growth by compounds 8m (red square), 8q (green triangle), 8r(blue diamond), 8p (dark triangle) and 2 (Apogossypol) (dark dots) inthe H460 human lung cell line. Cells were treated for 3 days and cellviability was evaluated using ATP-LITE assay. (D) Mouse embryonicfibroblast cells with wild-type (MEF/WT; blue bars) orbax^(−/−)bak^(−/−) double knockout (red bars) genotypes were treatedwith various 5,5′ substituted Apogossypol derivatives at 10 μM andapoptosis was monitored by Annexin V-FITC assays.

FIG. 15 shows: (A) Chemical stability of Apogossypol derivatives whenleft at room temperature in powder form: 8m (red dot), 8p (greensquare), 8q (purple dot), 8r (blue triangle), 8k (pink dot), 12e (darkdot), 2 (Apogossypol with ascorbic acid, dark square) and 2(Apogossypol, dark triangle). Chemical stability was evaluated in theair for 60 days at room temperature. The stability was monitored using acombination of HPLC and LCMS. (B) Effects of 5,5′ substitutedApogossypol derivatives on shrinkage of Bcl-2 mouse spleen at a singleintraperitoneal injection dose of 0.072 mmol/kg. All shrinkage data arepercentage of maximum reduction of mice spleen size. (C) % Weight lossin mice induced by single ip injection of various amount of compound 8r.(D) Effects of compound 8r at 42 mg/kg (0.06 mmol/kg) on reduction ofspleen weight of six Bcl-2 mice treatment with a single intraperitonealinjection. Data shown as means±S.E. (n=6). P<0.0001.

FIG. 16 shows a synthetic scheme that can be used to synthesize some ofthe compounds of the disclosure.

FIG. 17 shows a general synthetic scheme that can be used to synthesizesome of the compounds of the disclosure.

FIG. 18 shows a general synthetic scheme that can be used to synthesizesome of the compounds of the disclosure.

FIG. 19 shows the ITC studies of 5,5′ substituted Apogossypolderivatives.

FIG. 20 shows: (A) Compound 8r competes with the binding of Bcl-2 familyproteins to FITC-Bim BH3 peptide; (B) Cytotoxicity assays of ABT-737against BP3 using Annexin V-FITC and propidium iodide assay.

FIG. 21 shows the cytotoxicity assays of 5,5′ substituted Apogossypolderivatives against (A) BP3 cell and (B) RS11846 cancer cell lines usingAnnexin V-FITC and propidium iodide assay.

DETAILED DESCRIPTION OF THE DISCLOSURE

Unless otherwise defined, scientific and technical terms used inconnection with the disclosure shall have the meanings that are commonlyunderstood by those of ordinary skill in the art. Further, unlessotherwise required by context, singular terms shall include pluralitiesand plural terms shall include the singular. Generally, nomenclaturesutilized in connection with, and techniques of, cell and tissue culture,molecular biology, and protein and oligo- or polynucleotide chemistryand hybridization described herein are those well known and commonlyused in the art. Standard techniques are used for recombinant DNA,oligonucleotide synthesis, and tissue culture and transformation (e.g.,electroporation, lipofection). Enzymatic reactions and purificationtechniques are performed according to manufacturer's specifications oras commonly accomplished in the art or as described herein. Thenomenclatures utilized in connection with, and the laboratory proceduresand techniques of, analytical chemistry, synthetic organic chemistry,and medicinal and pharmaceutical chemistry described herein are thosewell known and commonly used in the art. Standard techniques are usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

The following terms, definitions and abbreviations further apply:

The term “patient” refers to organisms to be treated by the methods ofthe disclosure. Such organisms include, but are not limited to, humansand other mammals. In the context of the disclosure, the term “subject”generally refers to an individual who will receive or who has receivedtreatment described herein (e.g., administration of the compounds of thedisclosure, and optionally one or more additional therapeutic agents).

The term “BCL-2 family of proteins” refers to the family of proteinsthat currently includes at least the following six proteins: BCL-X_(L),BCL-2, BCL-W, BCL-B, BFL-1, and MCL-1.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e. unbranched) or branched chain,or cyclic hydrocarbon radical, or combination thereof, which may befully saturated, mono- or polyunsaturated and can include di- andmultivalent radicals, having the number of carbon atoms designated (i.e.C₁-C₁₀ means one to ten carbons). Examples of saturated hydrocarbonradicals include, but are not limited to, groups such as methyl, ethyl,n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. Alkyl groups which arelimited to hydrocarbon groups are termed “homoalkyl.”

Specific values listed herein for groups, substituents, and ranges, arefor illustration; they do not exclude other defined values or othervalues within defined ranges for the groups and substituents. Forexample, “alkyl” can be methyl, ethyl, propyl, isopropyl, butylisobutyl, sec-butyl, pentyl, 3-pentyl, or hexyl; cycloalkyl can becyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; “—O(C₁-C₆)alkyl(alkoxy)” can be methoxy, ethoxy, propoxy, isopropoxy, butoxy,iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkyl, as exemplified, but not limited,by —CH₂CH₂CH₂CH₂—, —CH₂CH═CHCH₂—, —CH₂C.ident.CCH₂—,—CH₂CH₂CH(CH₂CH₂CH₃)CH₂—. Typically, an alkyl (or alkylene) group willhave from 1 to 24 carbon atoms, which includes those groups having 10 orfewer carbon atoms. A “lower alkyl” or “lower alkylene” is a shorterchain alkyl or alkylene group, generally having eight or fewer carbonatoms.

The terms “alkyl, alkoxy, alkenyl, alkynyl,” etc. denote both straightand branched groups; but reference to an individual group such as“propyl” embraces the straight chain group, a branched chain isomer suchas “isopropyl” being specifically referred to.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of atleast one carbon atoms and at least one heteroatom selected from thegroup consisting of O, N, P, Si and S, and wherein the nitrogen,phosphorus, and sulfur atoms may optionally be oxidized and the nitrogenheteroatom may optionally be quaternized. The heteroatom(s) O, N, P andS and Si may be placed at any interior position of the heteroalkyl groupor at the position at which alkyl group is attached to the remainder ofthe molecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,—CH═CH—N(CH₃)—CH₃, O—CH₃, —O—CH₂—CH₃, and —CN. Up to two or threeheteroatoms may be consecutive, such as, for example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃. Similarly, the term “heteroalkylene” by itself or aspart of another substituent means a divalent radical derived fromheteroalkyl, as exemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxo,alkylenedioxo, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)OR′—includes both —C(O)OR′— and —R′OC(O)—. As described above, heteroalkylgroups, as used herein, include those groups that are attached to theremainder of the molecule through a heteroatom, such as —C(O)R′,—C(O)NR′, —NR′R′, —OR′, —SR′, and/or —SO₂R′. Where “heteroalkyl” isrecited, followed by recitations of specific heteroalkyl groups, such as—NR′R″ or the like, it will be understood that the terms heteroalkyl and—NR′R″ are not redundant or mutually exclusive. Rather, the specificheteroalkyl groups are recited to add clarity. Thus, the term“heteroalkyl” should not be interpreted herein as excluding specificheteroalkyl groups, such as —NR′R″ or the like.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, re, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. The terms “cycloalkylene”and “heterocycloalkylene” refer to the divalent derivatives ofcycloalkyl and heterocycloalkyl, respectively.

More specifically, the term “alkyl” refers to a branched or unbranchedsaturated hydrocarbon group of 1 to 6 carbon atoms, such as methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, and thelike. Alkyl groups herein contain 1 to 6 carbon atoms, such as, forexample, methyl, ethyl, and the like. As used herein the term “alkyl”also includes the term “cycloalkyl,” which refers to a cyclic alkylgroup of three to eight, including three, five or six, carbon atoms. Theterm “cycloalkylene” as used herein refers to a divalent cyclic alkylenegroup, typically a 3-, 5-, 6-, or 8-membered ring.

The term “alkoxy” as used herein refers to an alkyl group bound througha single, terminal ether linkage, i.e., an “alkoxy” group may be definedas —OR, where R is alkyl as defined herein. A “lower alkoxy” grouprefers to an alkoxy group containing 1 to 6, carbon atoms.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent which can be a single ring or multiplerings (from 1 to 3 rings) which are fused together or linked covalently.The term “heteroaryl” refers to aryl groups (or rings) that contain fromone to four heteroatoms (in each separate ring in the case of multiplerings) selected from N, O, and S, wherein the nitrogen and sulfur atomsare optionally oxidized, and the nitrogen atom(s) are optionallyquaternized. A heteroaryl group can be attached to the remainder of themolecule through a carbon or heteroatom. Non-limiting examples of aryland heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl,4-biphenyl, 1-pynolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 4-pyrimidyl,5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl,5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and6-quinolyl. Substituents for each of above noted aryl and heteroarylring systems are selected from the group of acceptable substituentsdescribed below. The terms “arylene” and “heteroarylene” refer to thedivalent radicals of aryl and heteroaryl, respectively.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxo, arylthioxo, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like). However, theterm “haloaryl,” as used herein is meant to cover aryls substituted withone or more halogens.

The term “aryl” as used herein refers to an aromatic carbocyclic ring,typically 6- or 10-membered, wherein at least one ring is aromatic. Forexample, “aryl” denotes a phenyl group or an ortho-fused bicycliccarbocyclic group having about nine to ten ring atoms in which at leastone ring is aromatic.

“Heteroaryl” encompasses a group attached via a ring carbon of amonocyclic aromatic ring containing five or six ring atoms consisting ofcarbon and one to four heteroatoms each independently may benon-peroxide oxygen, sulfur, and N(X), where X is absent or is H, O,(C₁-C₄)alkyl, phenyl or benzyl, as well as a group of an ortho-fusedbicyclic-heterocycle of about eight to ten ring atoms derived therefrom,particularly a benz-derivative or one derived by fusing a propylene,trimethylene, or tetramethylene digroup thereto.

Where a heteroalkyl, heterocycloalkyl, or heteroaryl includes a specificnumber of members (e.g. “3 to 7 membered”), the term “member” referrersto a carbon or heteroatom.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like. The term “halo” also refers to fluoro, chloro, bromo, or iodo.

The term “oxo” as used herein means an oxygen that is double bonded to acarbon atom.

Each of above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl, and“heterocycloalkyl”, “aryl,” “heteroaryl” as well as their divalentradical derivatives) are meant to include both substituted andunsubstituted forms of the indicated radical. Substituents for each typeof radical are provided below.

Substituents for alkyl, heteroalkyl, cycloalkyl, heterocycloalkylmonovalent and divalent derivative radicals (including those groupsoften referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl,alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —C(O)NR′R″, OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)OR′, —NR—C(NR′R″)=NR′″, —S(O)R′,—S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and —NO₂ in a number ranging fromzero to (2 m′+1), where m′ is the total number of carbon atoms in suchradical. R′, R″, R′″ and R″″ each independently refer to hydrogen,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl (e.g., aryl substituted with 1-3 halogens),substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, orarylalkyl groups. When a compound of the disclosure includes more thanone R group, for example, each of the R groups is independently selectedas are each R′, R″, R′″ and R″″ groups when more than one of thesegroups is. When R′ and R′″ are attached to the same nitrogen atom, theycan be combined with the nitrogen atom to form a 4-, 5-, 6-, or7-membered ring. For example, —NR′R″ is meant to include, but not belimited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussionof substituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH—, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for alkyl radicals above,exemplary substituents for aryl and heteroaryl groups (as well as theirdivalent derivatives) are varied and are selected from, for example:halogen, —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′,—CO₂R′, —C(O)NR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″,—NR″C(O)OR′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′,—S(O)₂NR′R″, —NRSO₂R′, —CN and —NO₂, —R′, —N₃, —CH(Ph)₂,fluoro(C₁-C₄)alkoxo, and fluoro(C₁-C₄)alkyl, in a number ranging fromzero to the total number of open valences on aromatic ring system; andwhere R′, R″, R′″ and R″″ are independently selected from hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl andsubstituted or unsubstituted heteroaryl. When a compound of thedisclosure includes more than one R′ group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is.

Two of the substituents on adjacent atoms of aryl or heteroaryl ring mayoptionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, wherein Tand U are independently —NR—, —O—, —CRR′— or a single bond, and q is aninteger of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂), —B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)₅—X′—(C″R′″)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″ and R′″ are independentlyselected from hydrogen, substituted or unsubstituted alkyl, substitutedor unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl.

As used herein, the term “heteroatom” or “ring heteroatom” is meant toinclude oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

An “aminoalkyl” as used herein refers to an amino group covalently boundto an alkylene linker. The amino group is —NR′R″, wherein R′ and R″ aretypically selected from hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.

A “substituent group,” as used herein, means a group selected from thefollowing moieties:

(A) —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

(B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, andheteroaryl, substituted with at least one substituent selected from:

(i) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

(ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, andheteroaryl, substituted with at least one substituent selected from: (a)oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

(b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, orheteroaryl, substituted with at least one substituent selected from oxo,—OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, and unsubstituted heteroaryl.

A “size-limited substituent” or “size-limited substituent group,” asused herein means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₄-C₈cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 4 to 8 membered heterocycloalkyl.

A “lower substituent” or “lower substituent group,” as used herein meansa group selected from all of the substituents described above for a“substituent group,” wherein each substituted or unsubstituted alkyl isa substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₅-C₇ cycloalkyl, and each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7membered heterocycloalkyl.

The compounds of the disclosure may exist as salts. The disclosureincludes such salts. Examples of applicable salt forms includehydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates,maleates, acetates, citrates, fumarates, tartrates (eg (+)-tartrates,(−)-tartrates or mixtures thereof including racemic mixtures,succinates, benzoates and salts with amino acids such as glutamic acid.These salts may be prepared by methods known to those skilled in art.Also included are base addition salts such as sodium, potassium,calcium, ammonium, organic amino, or magnesium salt, or a similar salt.When compounds of the disclosure contain relatively basicfunctionalities, acid addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredacid, either neat or in a suitable inert solvent. Examples of acceptableacid addition salts include those derived from inorganic acids likehydrochloric, hydrobromic, nitric, carbonic, monohydrogen-carbonic,phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,mono-hydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived organic acids like acetic, propionic,isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric,lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric,tartaric, methane-sulfonic, and the like. Also included are salts ofamino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like. Certain specificcompounds of the disclosure contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts.

The neutral forms of the compounds are regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents.

Certain compounds of the disclosure can exist in unsolvated forms aswell as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the disclosure. Certain compounds of the disclosuremay exist in multiple crystalline or amorphous forms. In general, allphysical forms are equivalent for the uses contemplated by thedisclosure and are intended to be within the scope of the disclosure.

Certain compounds of the disclosure possess asymmetric carbon atoms(optical or chiral centers) or double bonds; the enantiomers, racemates,diastereomers, tautomers, geometric isomers, stereoisometric forms thatmay be defined, in terms of absolute stereochemistry, as (R)- or (S)-or, as (D)- or (L)- for amino acids, and individual isomers areencompassed within the scope of the disclosure. The compounds of thedisclosure do not include those which are known in art to be toounstable to synthesize and/or isolate. The disclosure is meant toinclude compounds in racemic and optically pure forms. Optically active(R)- and (S)-, or (D)- and (L)-somers may be prepared using chiralsynthons or chiral reagents, or resolved using conventional techniques.When the compounds described herein contain olefinic bonds or othercenters of geometric asymmetry, and unless specified otherwise, it isintended that the compounds include both E and Z geometric isomers.

The term “tautomer,” as used herein, refers to one of two or morestructural isomers which exist in equilibrium and which are readilyconverted from one isomeric form to another.

It will be apparent to one skilled in the art that certain compounds ofthis disclosure may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the disclosure.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the compounds are within the scope of the disclosure.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ in the presence of one or moreisotopically enriched atoms. For example, compounds having thestructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbonare within the scope of this disclosure.

The compounds of the disclosure may also contain unnatural proportionsof atomic isotopes at one or more of atoms that constitute suchcompounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (3H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe disclosure, whether radioactive or not, are encompassed within thescope of the disclosure.

The term “pharmaceutically acceptable salts” is meant to include saltsof active compounds which are prepared with relatively nontoxic acids orbases, depending on the particular substituent moieties found on thecompounds described herein. When compounds of the disclosure containrelatively acidic functionalities, base addition salts can be obtainedby contacting the neutral form of such compounds with a sufficientamount of the desired base, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium salt,or a similar salt. When compounds of the disclosure contain relativelybasic functionalities, acid addition salts can be obtained by contactingthe neutral form of such compounds with a sufficient amount of thedesired acid, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable acid addition salts include those derivedfrom inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived fromrelatively nontoxic organic acids like acetic, propionic, isobutyric,maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,methanesulfonic, and the like. Also included are salts of amino acidssuch as arginate and the like, and salts of organic acids likeglucuronic or galactunoric acids and the like (see, for example, Bergeet al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977,66, 1-19). Certain specific compounds of the disclosure contain bothbasic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts.

In addition to salt forms, the disclosure provides compounds, which arein a prodrug form. Prodrugs of the compounds described herein are thosecompounds that readily undergo chemical changes under physiologicalconditions to provide the compounds of the disclosure. Additionally,prodrugs can be converted to the compounds of the disclosure by chemicalor biochemical methods in an ex vivo environment. For example, prodrugscan be slowly converted to the compounds of the disclosure when placedin a transdermal patch reservoir with a suitable enzyme or chemicalreagent.

The terms “a,” “an,” or “a(n)”, when used in reference to a group ofsubstituents herein, mean at least one. For example, where a compound issubstituted with “an” alkyl or aryl, the compound is optionallysubstituted with at least one alkyl and/or at least one aryl. Moreover,where a moiety is substituted with an R substituent, the group may bereferred to as “R-substituted.” Where a moiety is R-substituted, themoiety is substituted with at least one R substituent and each Rsubstituent is optionally different.

Description of compounds of the disclosure are limited by principles ofchemical bonding known to those skilled in the art. Accordingly, where agroup may be substituted by one or more of a number of substituents,such substitutions are selected so as to comply with principles ofchemical bonding and to give compounds which are not inherently unstableand/or would be known to one of ordinary skill in the art as likely tobe unstable under ambient conditions, such as aqueous, neutral, andseveral known physiological conditions. For example, a heterocycloalkylor heteroaryl is attached to the remainder of the molecule via a ringheteroatom in compliance with principles of chemical bonding known tothose skilled in the art thereby avoiding inherently unstable compounds.

The terms “treating” or “treatment” in reference to a particular diseaseincludes prevention of the disease.

The term “prodrug” or “pro-drug” refers to an agent that is convertedinto the parent drug in vivo. Prodrugs are often useful because, in somesituations, they may be easier to administer than the parent drug. Theymay, for instance, be bioavailable by oral administration whereas theparent is not. The prodrug may also have improved solubility inpharmaceutical compositions over the parent drug, or may demonstrateincreased palatability or be easier to formulate.

As used herein, the term “apogossypol” is a broad term which includes,without limitation, L-apogossypol, D-apogossypol, racemic apogossypol,S-apogossypol, R-apogossypol, (−) apogossypol and (+) apogossypol, andincludes (−)apogossypol that is substantially free of (+)apogossypol.

Throughout the disclosure, when a particular compound is mentioned byname, for example, apogossypol, it is understood that the scope of thedisclosure encompasses pharmaceutically acceptable salts, esters,amides, metabolites, or prodrugs of the named compound.

It will be appreciated by those skilled in the art that compounds of thedisclosure having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the disclosure encompasses anyracemic, optically active, polymorphic, or stereoisomeric form, ormixtures thereof, of a compound of the disclosure, which possesses theuseful properties described herein. Also, if the named compoundcomprises a chiral center, the scope of the disclosure also includescompositions comprising the racemic mixture of the two enantiomers, aswell as compositions comprising each enantiomer individually,substantially free of the other enantiomer. Thus, for example,contemplated herein is a composition comprising the S enantiomersubstantially free of the R enantiomer, or a composition comprising theR enantiomer substantially free of the S enantiomer.

By “substantially free” it is meant that the composition comprises lessthan 10%, or less than 8%, or less than 5%, or less than 3%, or lessthan 1% of the minor enantiomer. If the named compound comprises morethan one chiral center, the scope of the disclosure also includescompositions comprising a mixture of the various diastereomers, as wellas compositions comprising each diastereomer substantially free of theother diastereomers. Thus, for example, commercially availableapogossypol is a racemic mixture comprising two separate enantiomers.The recitation of “apogossypol” throughout this disclosure includescompositions that comprise the racemic mixture of apogossypol,compositions that comprise the (+) enantiomer substantially free of the(−) enantiomer, and compositions that comprise the (−) enantiomersubstantially free of the (+) enantiomer.

It is well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase) and how to determine the anti cancer activity usingthe standard tests described herein, or using other similar tests whichare well known in the art.

The term “pharmaceutical composition” refers to a mixture of a compoundwith other chemical components, such as diluents or carriers. Thepharmaceutical composition facilitates administration of the compound toan organism. Multiple techniques of administering a compound exist inthe art including, but not limited to, oral, injection, aerosol,parenteral, and topical administration. Pharmaceutical compositions canalso be obtained by reacting compounds with inorganic or organic acidssuch as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like.

The term “pharmaceutically acceptable salt” refers to a formulation of acompound that does not cause significant irritation to an organism towhich it is administered and does not abrogate the biological activityand properties of the compound. Pharmaceutical salts can be obtained byreacting a compound of the disclosure with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like. Pharmaceuticalsalts can also be obtained by reacting a compound of the disclosure witha base to form a salt such as an ammonium salt, an alkali metal salt,such as a sodium or a potassium salt, an alkaline earth metal salt, suchas a calcium or a magnesium salt, a salt of organic bases such asdicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine,and salts thereof with amino acids such as arginine, lysine, and thelike.

“Inflammation” as used herein is a general term for the localaccumulation of fluid, plasma proteins, and white blood cells that isinitiated by physical injury, infection, or a local immune response.Many different forms of inflammation are associated with differentdiseases. “Inflammation-associated” diseases include, for example,lupus, psoriasis, rheumatoid arthritis, and inflammatory bowel disease.Other inflammation-associated diseases are discussed herein.

As used herein, the terms “anti-inflammatory agent” refers to anyanti-inflammatory compounds that are used in the treatment ofinflammation.

“Treatment,” as used herein, pertains to the therapeutic administrationof the compounds of the disclosure for the prevention, amelioration, orcure of disease.

The term “pharmaceutical agent or drug” as used herein refers to achemical compound or composition capable of inducing a desiredtherapeutic effect when properly administered to a patient.

As used herein, “substantially pure” means an object species is thepredominant species (i.e., on a molar basis it is more abundant than anyother individual species in the composition), and a substantiallypurified fraction is a composition wherein the object species comprisesat least about 50 percent (on a molar basis) of all macromolecularspecies. Generally, a substantially pure composition will comprise morethan about 80 percent of all macromolecular species in the composition,for example, more than about 85%, 90%, 95%, and 99%. The object speciesmay be also purified to essential homogeneity (contaminant speciescannot be detected in the composition by conventional detectionmethods), wherein the composition consists essentially of a singlespecies.

In one aspect the disclosure provides a compound having structure A, ora pharmaceutically acceptable salt, hydrate, N-oxide, or solvatethereof:

wherein each R is independently H, C(O)X, C(O)NHX, NH(CO)X, SO₂NHX, orNHSO₂X; and X is hydroxyl, alkyl, substituted alkyl, aryl, substitutedaryl, alkylaryl, substituted alkylaryl, heterocycle, or substitutedheterocycle.

In another aspect the disclosure provides a compound having structure A,wherein each R is independently NH(CO)X; and X is alkyl, substitutedalkyl, aryl, substituted aryl, alkylaryl, substituted alkylaryl,heterocycle, or substituted heterocycle.

In another aspect the disclosure provides a compound having structure A,wherein X is (C₁-C₆)alkyl, substituted (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl,substituted (C₃-C₈)cycloalkyl, phenyl, substituted phenyl,(C₁-C₆)alkylaryl or substituted (C₁-C₆)alkylaryl, wherein eachsubstitutent is (C₁-C₆)alkyl, trifluoromethyl, halogen, phenyl orphenoxy.

In another aspect the disclosure provides a compound having structure A,wherein each R is independently

In another aspect the disclosure provides a compound having structure A,wherein each R is independently C(O)NHCH₂CH(CH₃)C₆H₅.

In another aspect the disclosure provides a compound having structure A,wherein X is an alkylaryl.

In another aspect the disclosure provides a compound having structure A,wherein X is benzyl.

In another aspect the disclosure provides a compound having structure A,wherein the compound is:

In another aspect the disclosure provides a compound having structure A,wherein the compound is compound I-XXII:

In another aspect the disclosure provides a compound having structure A,wherein the compound is compound I:

In another aspect the disclosure provides a compound having structure A,wherein the compound is compound XXI:

In another aspect the disclosure provides a compound having structure A,wherein the compound is compound XXII:

In another aspect the disclosure provides methods for treating a diseaseor a disorder, by administering to a subject in need thereof atherapeutically effective amount of a compound having structure A, or acombination thereof, or a pharmaceutically acceptable salt, hydrate,N-oxide, or solvate thereof, thereby treating the disease or thedisorder.

In another aspect the disclosure provides methods for treating a diseaseor a disorder, by administering to a subject in need thereof atherapeutically effective amount of a compound having structure A,wherein the disease or the disorder is cancer.

In another aspect the disclosure provides methods for treating a diseaseor a disorder, by administering to a subject in need thereof atherapeutically effective amount of a compound having structure A,wherein the disease or the disorder is cancer, and wherein cancer islung cancer, breast cancer, prostate cancer, or lymphomas.

In another aspect the disclosure provides methods for treating a diseaseor a disorder, by administering to a subject in need thereof atherapeutically effective amount of a compound having structure A,wherein the treatment includes inhibition of activity of at least oneBCL-2 family protein.

In another aspect the disclosure provides methods for treating a diseaseor a disorder, by administering to a subject in need thereof atherapeutically effective amount of a compound having structure A, byadministering the compound having structure A in combination with ananti-cancer agent.

In another aspect the disclosure provides methods for treating cancer oran autoimmune disease in a subject having at least one elevated BCL-2family protein expression level by administering to the subject atherapeutically effective amount of a compound having structure A, or acombination thereof, or a pharmaceutically acceptable salt, hydrate,N-oxide, or solvate thereof.

In another aspect the disclosure provides methods for treating cancer oran autoimmune disease in a subject having at least one elevated BCL-2family protein expression level by administering to the subject atherapeutically effective amount of a compound having structure A, bydetermining whether the subject is responsive to a therapy that utilizesthe compound, and determining the level of at least one of the BCL-2family protein in the subject and comparing to a normal control sample,wherein an elevated level is indicative of a subject responsive to thetherapy that the compound, or a pharmaceutically acceptable salt,hydrate, N-oxide, or solvate thereof.

In another aspect the disclosure provides methods for treating cancer oran autoimmune disease in a subject having at least one elevated BCL-2family protein expression level comprising administering to the subjecta therapeutically effective amount of a compound having structure A, bydetermining whether the subject is responsive to a therapy that utilizesthe compound, and determining the level of at least one of the BCL-2family protein in the subject and comparing to a normal control sample,wherein an elevated level is indicative of a subject responsive to thetherapy that the compound, or a pharmaceutically acceptable salt,hydrate, N-oxide, or solvate thereof, wherein the determination is madebased on a sample from the subject.

In another aspect the disclosure provides methods for determiningwhether a subject is responsive to a therapy that utilizes of a compoundhaving structure A, or a combination thereof, or a pharmaceuticallyacceptable salt, hydrate, N-oxide, or solvate thereof, by determiningthe level of at least one of the BCL-2 family protein in the subject andcomparing to a normal control sample, wherein an elevated level isindicative of a subject responsive to the therapy that utilizes thecompound, or a pharmaceutically acceptable salt, hydrate, N-oxide, orsolvate thereof.

In another aspect the disclosure provides methods for determiningwhether a subject is responsive to a therapy that utilizes of a compoundhaving structure A, or a combination thereof, or a pharmaceuticallyacceptable salt, hydrate, N-oxide, or solvate thereof, by determiningthe level of at least one of the BCL-2 family protein in the subject andcomparing to a normal control sample, wherein an elevated level isindicative of a subject responsive to the therapy that utilizes thecompound, or a pharmaceutically acceptable salt, hydrate, N-oxide, orsolvate thereof, and wherein the determination is made based on a samplefrom the subject.

In another aspect the disclosure provides methods for determiningwhether a subject is responsive to a therapy that utilizes of a compoundhaving structure A, or a combination thereof, or a pharmaceuticallyacceptable salt, hydrate, N-oxide, or solvate thereof, by determiningthe level of at least one of the BCL-2 family protein in the subject andcomparing to a normal control sample, wherein an elevated level isindicative of a subject responsive to the therapy that utilizes thecompound, or a pharmaceutically acceptable salt, hydrate, N-oxide, orsolvate thereof, and wherein the sample is a biological fluid or tumorsample.

In another aspect the disclosure provides methods for determiningwhether a subject is responsive to a therapy that utilizes of a compoundhaving structure A, or a combination thereof, or a pharmaceuticallyacceptable salt, hydrate, N-oxide, or solvate thereof, by determiningthe level of at least one of the BCL-2 family protein in the subject andcomparing to a normal control sample, wherein an elevated level isindicative of a subject responsive to the therapy that utilizes thecompound, or a pharmaceutically acceptable salt, hydrate, N-oxide, orsolvate thereof, and wherein the BCL-2 family polynucleotide orpolypeptide is selected from BCL-2, BCL-XL, BCL-W, MCL-1, and BCL-A1.

In another aspect the disclosure provides methods for inducing apoptosisin a cell having a level of at least one of the BCL-2 family proteinmember greater than levels in a control cell, by administering to thecell an effective amount of a compound having structure A, or acombination thereof, or a pharmaceutically acceptable salt, hydrate,N-oxide, or solvate thereof, to reduce the level of BCL-2 familyprotein(s) and induce apoptosis in the cell.

In another aspect the disclosure provides methods for inducing apoptosisin a cell having a level of at least one of the BCL-2 family proteinmember greater than levels in a control cell, by administering to thecell an effective amount of a compound having structure A, or acombination thereof, or a pharmaceutically acceptable salt, hydrate,N-oxide, or solvate thereof, to reduce the level of BCL-2 familyprotein(s) and induce apoptosis in the cell, wherein the cell is acancer cell.

In another aspect the disclosure provides methods for inducing apoptosisin a cell having a level of at least one of the BCL-2 family proteinmember greater than levels in a control cell, by administering to thecell an effective amount of a compound having structure A, or acombination thereof, or a pharmaceutically acceptable salt, hydrate,N-oxide, or solvate thereof, to reduce the level of BCL-2 familyprotein(s) and induce apoptosis in the cell, wherein the cell is acancer cell, and wherein cancer is lung cancer, breast cancer, prostatecancer, or lymphomas.

In another aspect the disclosure provides methods for inducing apoptosisin a cell having a level of at least one of the BCL-2 family proteinmember greater than levels in a control cell, by administering to thecell an effective amount of a compound having structure A, or acombination thereof, or a pharmaceutically acceptable salt, hydrate,N-oxide, or solvate thereof, to reduce the level of BCL-2 familyprotein(s) and induce apoptosis in the cell, wherein the cell is a cellof the immune system.

In another aspect the disclosure provides methods for determining theeffectiveness of a therapeutic regimen including administration of thecompound of a compound having structure A, or a combination thereof, ora pharmaceutically acceptable salt, hydrate, N-oxide, or solvatethereof, in a subject by comparing the level of a BCL-2 family proteinin a cell of the subject prior to and during treatment with thecompound, or a pharmaceutically acceptable salt, hydrate, N-oxide, orsolvate thereof, wherein a decreased level of BCL-2 family protein isindicative of effectiveness of the therapy that utilizes the compound,or a pharmaceutically acceptable salt, hydrate, N-oxide, or solvatethereof.

In another aspect the disclosure provides methods for determining theeffectiveness of a therapeutic regimen including administration of thecompound of a compound having structure A, or a combination thereof, ora pharmaceutically acceptable salt, hydrate, N-oxide, or solvatethereof, in a subject by comparing the level of a BCL-2 family proteinin a cell of the subject prior to and during treatment with thecompound, or a pharmaceutically acceptable salt, hydrate, N-oxide, orsolvate thereof, wherein a decreased level of BCL-2 family protein isindicative of effectiveness of the therapy that utilizes the compound,or a pharmaceutically acceptable salt, hydrate, N-oxide, or solvatethereof, wherein the subject has cancer.

In another aspect the disclosure provides methods for determining theeffectiveness of a therapeutic regimen including administration of thecompound of a compound having structure A, or a combination thereof, ora pharmaceutically acceptable salt, hydrate, N-oxide, or solvatethereof, in a subject by comparing the level of a BCL-2 family proteinin a cell of the subject prior to and during treatment with thecompound, or a pharmaceutically acceptable salt, hydrate, N-oxide, orsolvate thereof, wherein a decreased level of BCL-2 family protein isindicative of effectiveness of the therapy that utilizes the compound,or a pharmaceutically acceptable salt, hydrate, N-oxide, or solvatethereof, wherein the subject has cancer, and wherein cancer is lungcancer, breast cancer, prostate cancer, or lymphomas.

In another aspect the disclosure provides methods for determining theeffectiveness of a therapeutic regimen including administration of thecompound of a compound having structure A, or a combination thereof, ora pharmaceutically acceptable salt, hydrate, N-oxide, or solvatethereof, in a subject by comparing the level of a BCL-2 family proteinin a cell of the subject prior to and during treatment with thecompound, or a pharmaceutically acceptable salt, hydrate, N-oxide, orsolvate thereof, wherein a decreased level of BCL-2 family protein isindicative of effectiveness of the therapy that utilizes the compound,or a pharmaceutically acceptable salt, hydrate, N-oxide, or solvatethereof, wherein the subject has an autoimmune disorder.

In another aspect the disclosure provides methods for treatinginflammation in a subject by administering to the subject in need of thetreatment a pharmaceutically effective amount of a compound havingstructure B:

wherein each of R⁶, R⁸, R⁹ and R¹⁰ is hydrogen, hydroxyl, —(C₁-C₆)alkyl,—O(C₁-C₆)alkyl, —(C₁-C₆)alkylhalo, —OC(O)(C₁-C₆)alkyl, or halo; and eachR⁷ is independently hydrogen, —(C₁-C₆)alkyl, —(C₃-C₈)cycloalkyl,—(C₆-C₁₀)aryl, and —(C₁-C₆)alkyl(C₆-C₁₀)aryl, C(O)X, C(O)NHX, NH(CO)X,SO₂NHX, and NHSO₂X, wherein X is alkyl, substituted alkyl, aryl,substituted aryl, alkylaryl, substituted alkylaryl, heterocycle, or asubstituted heterocycle, or a pharmaceutically acceptable salt, hydrate,N-oxide, or solvate thereof, to reduce the inflammation thereby.

In another aspect the disclosure provides methods for treatinginflammation in a subject by administering to the subject in need of thetreatment a pharmaceutically effective amount of a compound havingstructure B, wherein the compound is apogossypol.

In another aspect the disclosure provides methods for treatinginflammation in a subject by administering to the subject in need of thetreatment a pharmaceutically effective amount of a compound havingstructure B, wherein the compound is apogossypol, wherein the compoundis (−) apogossypol substantially free of (+) apogossypol.

In another aspect the disclosure provides methods for treatinginflammation in a subject by administering to the subject in need of thetreatment a pharmaceutically effective amount of a compound havingstructure B, wherein each of R⁶, R⁸, R⁹ and R¹⁰ is independentlyhydrogen, —OH, —OCH₃, —CF₃, —CH₃, —OC₂H₅, —OC(O)CH₃, F, Cl, or Br.

In another aspect the disclosure provides methods for treatinginflammation in a subject by administering to the subject in need of thetreatment a pharmaceutically effective amount of a compound havingstructure B, wherein each R⁷ is independently hydrogen, ethyl, n-propyl,iso-propyl, n-butyl, tent-butyl, iso-butyl, sec-butyl, or cyclohexyl.

In another aspect the disclosure provides methods for treatinginflammation in a subject by administering to the subject in need of thetreatment a pharmaceutically effective amount of a compound havingstructure B, wherein each R¹⁰ is independently hydrogen, —OH, —OCH₃,—CF₃, —CH₃, —OC₂H₅, —OC(O)CH₃, F, Cl, or Br.

In another aspect the disclosure provides methods for treatinginflammation in a subject by administering to the subject in need of thetreatment a pharmaceutically effective amount of a compound havingstructure B, wherein each R⁶, R⁸, and R⁹ is —OC(O)CH₃, each R⁷ isiso-propyl; and each R¹⁰ is —CH₃.

In another aspect the disclosure provides methods for treatinginflammation in a subject by administering to the subject in need of thetreatment a pharmaceutically effective amount of a compound havingstructure B, wherein the compound is a pro-drug of apogossypol.

In another aspect the disclosure provides methods for treatinginflammation in a subject by administering to the subject in need of thetreatment a pharmaceutically effective amount of a compound havingstructure B, wherein the compound is compound XXI:

In another aspect the disclosure provides methods for treatinginflammation in a subject by administering to the subject in need of thetreatment a pharmaceutically effective amount of a compound havingstructure B, wherein the compound is compound XXII:

In another aspect the disclosure provides methods for treatinginflammation in a subject by administering to the subject in need of thetreatment a pharmaceutically effective amount of a compound havingstructure B, wherein the subject is afflicted with a condition whereinthe condition is lupus erythmatosus, psoriasis, psoriatic arthritis,lupus nephritis, rheumatoid arthritis, multiple sclerosis, ulcerativecolitis, myasthenia gravis, ITP, TTP, Grave's disease, Hashimoto'sthyroiditis, Crohn's disease, autoimmune hemolytic anemias, insulindependent diabetes mellitus, glomerulonephritis, rheumatic fever,osteoarthritis, gouty arthritis, dermatitis, bronchitis, rhinitis,asthma, Sjogrens' syndrome, meningitis, adrenoleukodystrophy, CNSvasculitis, mitochondrial myopathies, Amyotrophic Lateral Sclerosis,Alzheimer's disease, or a tumor.

In another aspect the disclosure provides methods for treatinginflammation in a subject by administering to the subject in need of thetreatment a pharmaceutically effective amount of a compound havingstructure B, wherein the mitochondrial myopathy is MELAS syndrome, MERFsyndrome, Leber's disease, Wernicke's encephalopathy, Rett syndrome,homocystinuria, hyperprolinemia, nonketotic hyperglycinemia,hydroxybutyric aminoaciduria, sulfite oxidase deficiency, or combinedsystems disease (B12 deficiency).

In another aspect the disclosure provides methods for treatinginflammation in a subject by administering to the subject in need of thetreatment a pharmaceutically effective amount of a compound havingstructure B, and administering a selective serotonin reuptake inhibitor(SSRI).

In another aspect the disclosure provides methods for treatinginflammation in a subject in need of such treatment by administering tothe subject in need of the treatment an anti-inflammatory agent, whereinthe anti-inflammatory agent is gossypol, apogossypol, L-apogossypol,derivatives of apogossypol, theaflavin, theaflavin-3′-gallate,theaflavanin, (−) gallocatechin-3-gallate (GCG), (−)epigallocatechin-3-gallate (EGCG), (−) catechin-3-gallate (CG), (−)epicatechin-3-gallate (ECG), or derivatives of purpurogallin, to treatthe inflammation thereby.

In another aspect the disclosure provides methods for treatinginflammation in a subject in need of such treatment by administering tothe subject in need of the treatment an anti-inflammatory agent, whereinthe anti-inflammatory agent is gossypol, apogossypol, L-apogossypol,derivatives of apogossypol, theaflavin, theaflavin-3′-gallate,theaflavanin, (−) gallocatechin-3-gallate (GCG), (−)epigallocatechin-3-gallate (EGCG), (−) catechin-3-gallate (CG), (−)epicatechin-3-gallate (ECG), or derivatives of purpurogallin, to treatthe inflammation thereby, and administering a selective serotoninreuptake inhibitor (SSRI).

In another aspect the disclosure provides methods for treatinginflammation in a subject in need of such treatment by administering tothe subject in need of the treatment an anti-inflammatory agent, whereinthe anti-inflammatory agent is apogossypol.

In another aspect the disclosure provides methods for treatinginflammation in a subject in need of such treatment by administering tothe subject in need of the treatment an anti-inflammatory agent, whereinthe anti-inflammatory agent is (−) apogossypol substantially free of (+)apogossypol.

In another aspect the disclosure provides methods for treatinginflammation in a subject in need of such treatment by administering tothe subject in need of the treatment an anti-inflammatory agent, whereinthe anti-inflammatory agent is a derivative of apogossypol, wherein thederivative of apogossypol is a compound having structure B, wherein thederivative a purpurogallin derivative is 5D1, 1163, or 1142.

In another aspect the disclosure provides methods for treatinginflammation in a subject in need of such treatment by administering tothe subject in need of the treatment an anti-inflammatory agent, whereinthe anti-inflammatory agent is gossypol, apogossypol, L-apogossypol,derivatives of apogossypol, theaflavin, theaflavin-3′-gallate,theaflavanin, (−) gallocatechin-3-gallate (GCG), (−)epigallocatechin-3-gallate (EGCG), (−) catechin-3-gallate (CG), (−)epicatechin-3-gallate (ECG), or derivatives of purpurogallin, to treatthe inflammation thereby, wherein the inflammation is inflammationassociated with a condition wherein the condition is lupus erythmatosus,psoriasis, psoriatic arthritis, lupus nephritis, rheumatoid arthritis,multiple sclerosis, ulcerative colitis, myasthenia gravis, ITP, TTP,Grave's disease, Hashimoto's thyroiditis, Crohn's disease, autoimmunehemolytic anemias, insulin dependent diabetes mellitus,glomerulonephritis, rheumatic fever, osteoarthritis, gouty arthritis,dermatitis, bronchitis, rhinitis, asthma, Sjogren' syndrome, meningitis,adrenoleukodystrophy, CNS vasculitis, mitochondrial myopathies,Amyotrophic Lateral Sclerosis, Alzheimer's disease, or a tumor.

In another aspect the disclosure provides methods for treatinginflammation in a subject in need of such treatment by administering tothe subject in need of the treatment an anti-inflammatory agent, whereinthe anti-inflammatory agent is gossypol, apogossypol, L-apogossypol,derivatives of apogossypol, theaflavin, theaflavin-3′-gallate,theaflavanin, (−) gallocatechin-3-gallate (GCG), (−)epigallocatechin-3-gallate (EGCG), (−) catechin-3-gallate (CG), (−)epicatechin-3-gallate (ECG), or derivatives of purpurogallin, to treatthe inflammation thereby, wherein the inflammation is inflammationassociated with a condition wherein the condition is lupus erythmatosus,psoriasis, psoriatic arthritis, lupus nephritis, rheumatoid arthritis,multiple sclerosis, ulcerative colitis, myasthenia gravis, ITP, TTP,Grave's disease, Hashimoto's thyroiditis, Crohn's disease, autoimmunehemolytic anemias, insulin dependent diabetes mellitus,glomerulonephritis, rheumatic fever, osteoarthritis, gouty arthritis,dermatitis, bronchitis, rhinitis, asthma, Sjogren' syndrome, meningitis,adrenoleukodystrophy, CNS vasculitis, mitochondrial myopathies,Amyotrophic Lateral Sclerosis, Alzheimer's disease, or a tumor, whereinthe mitochondrial myopathy is MELAS syndrome, MERF syndrome, Leber'sdisease, Wernicke's encephalopathy, Rett syndrome, homocystinuria,hyperprolinemia, nonketotic hyperglycinemia, hydroxybutyricaminoaciduria, sulfite oxidase deficiency, or combined systems disease(B12 deficiency).

In another aspect the disclosure provides methods for treating a diseaseor a disorder, by administering to a subject in need thereof atherapeutically effective amount of a compound or stereoisomer thereofas described herein, or a combination thereof, or a pharmaceuticallyacceptable salt, hydrate, N-oxide, or solvate thereof, thereby treatingthe disease or the disorder.

In another aspect the disclosure provides methods for treating a diseaseor a disorder, by administering to a subject in need thereof atherapeutically effective amount of a compound or stereoisomer thereofas described herein, or a combination thereof, or a pharmaceuticallyacceptable salt, hydrate, N-oxide, or solvate thereof, thereby treatingthe disease or the disorder, wherein the disease or the disorder iscancer.

In another aspect the disclosure provides methods for treating a diseaseor a disorder, by administering to a subject in need thereof atherapeutically effective amount of a compound or stereoisomer thereofas described herein, or a combination thereof, or a pharmaceuticallyacceptable salt, hydrate, N-oxide, or solvate thereof, thereby treatingthe disease or the disorder, wherein the disease or the disorder iscancer, wherein cancer is lung cancer, breast cancer, prostate cancer,or lymphomas.

In another aspect the disclosure provides methods for treating a diseaseor a disorder, by administering to a subject in need thereof atherapeutically effective amount of a compound or stereoisomer thereofas described herein, or a combination thereof, or a pharmaceuticallyacceptable salt, hydrate, N-oxide, or solvate thereof, thereby treatingthe disease or the disorder, and wherein the treatment includesinhibition of activity of at least one BCL-2 family protein.

In another aspect the disclosure provides methods for treating a diseaseor a disorder, by administering to a subject in need thereof atherapeutically effective amount of a compound or stereoisomer thereofas described herein, or a combination thereof, or a pharmaceuticallyacceptable salt, hydrate, N-oxide, or solvate thereof, thereby treatingthe disease or the disorder, by administering the compound incombination with an anti-cancer agent.

In another aspect the disclosure provides methods for treating cancer oran autoimmune disease in a subject having at least one elevated BCL-2family protein expression level by administering to the subject atherapeutically effective amount of a compound or stereoisomer thereofas described herein, or a combination thereof, or a pharmaceuticallyacceptable salt, hydrate, N-oxide, or solvate thereof.

In another aspect the disclosure provides methods for treating cancer oran autoimmune disease in a subject having at least one elevated BCL-2family protein expression level by administering to the subject atherapeutically effective amount of a compound or stereoisomer thereofas described herein, or a combination thereof, or a pharmaceuticallyacceptable salt, hydrate, N-oxide, or solvate thereof, by determiningwhether the subject is responsive to a therapy that utilizes thecompound, by determining the level of at least one of the BCL-2 familyprotein in the subject and comparing to a normal control sample, whereinan elevated level is indicative of a subject responsive to the therapythat the compound, or a pharmaceutically acceptable salt, hydrate,N-oxide, or solvate thereof.

In another aspect the disclosure provides methods for treating cancer oran autoimmune disease in a subject having at least one elevated BCL-2family protein expression level by administering to the subject atherapeutically effective amount of a compound or stereoisomer thereofas described herein, or a combination thereof, or a pharmaceuticallyacceptable salt, hydrate, N-oxide, or solvate thereof, by determiningwhether the subject is responsive to a therapy that utilizes thecompound, by determining the level of at least one of the BCL-2 familyprotein in the subject and comparing to a normal control sample, whereinan elevated level is indicative of a subject responsive to the therapythat the compound, or a pharmaceutically acceptable salt, hydrate,N-oxide, or solvate thereof, wherein the determination is made based ona sample from the subject.

In another aspect the disclosure provides methods for determiningwhether a subject is responsive to a therapy that utilizes a compound orstereoisomer thereof as described herein, or a combination thereof, or apharmaceutically acceptable salt, hydrate, N-oxide, or solvate thereof,by determining the level of at least one of the BCL-2 family protein inthe subject and comparing to a normal control sample, wherein anelevated level is indicative of a subject responsive to the therapy thatutilizes the compound, or a pharmaceutically acceptable salt, hydrate,N-oxide, or solvate thereof.

In another aspect the disclosure provides methods for determiningwhether a subject is responsive to a therapy that utilizes a compound orstereoisomer thereof as described herein, or a combination thereof, or apharmaceutically acceptable salt, hydrate, N-oxide, or solvate thereof,by determining the level of at least one of the BCL-2 family protein inthe subject and comparing to a normal control sample, wherein anelevated level is indicative of a subject responsive to the therapy thatutilizes the compound, or a pharmaceutically acceptable salt, hydrate,N-oxide, or solvate thereof, wherein the determination is made based ona sample from the subject.

In another aspect the disclosure provides methods for determiningwhether a subject is responsive to a therapy that utilizes a compound orstereoisomer thereof as described herein, or a combination thereof, or apharmaceutically acceptable salt, hydrate, N-oxide, or solvate thereof,by determining the level of at least one of the BCL-2 family protein inthe subject and comparing to a normal control sample, wherein anelevated level is indicative of a subject responsive to the therapy thatutilizes the compound, or a pharmaceutically acceptable salt, hydrate,N-oxide, or solvate thereof, wherein the sample is a biological fluid ortumor sample.

In another aspect the disclosure provides methods for determiningwhether a subject is responsive to a therapy that utilizes a compound orstereoisomer thereof as described herein, or a combination thereof, or apharmaceutically acceptable salt, hydrate, N-oxide, or solvate thereof,by determining the level of at least one of the BCL-2 family protein inthe subject and comparing to a normal control sample, wherein anelevated level is indicative of a subject responsive to the therapy thatutilizes the compound, or a pharmaceutically acceptable salt, hydrate,N-oxide, or solvate thereof, wherein the BCL-2 family polynucleotide orpolypeptide is selected from BCL-2, BCL-XL, BCL-W, MCL-1, and BCL-A1.

In another aspect the disclosure provides methods for inducing apoptosisin a cell having a level of at least one of the BCL-2 family proteinmember greater than levels in a control cell, by administering to thecell an effective amount of a compound of structure A, or a combinationthereof, or a pharmaceutically acceptable salt, hydrate, N-oxide, orsolvate thereof, to reduce the level of BCL-2 family protein(s) andinduce apoptosis in the cell.

In another aspect the disclosure provides methods for inducing apoptosisin a cell having a level of at least one of the BCL-2 family proteinmember greater than levels in a control cell, by administering to thecell an effective amount of a compound having structure A, or acombination thereof, or a pharmaceutically acceptable salt, hydrate,N-oxide, or solvate thereof, to reduce the level of BCL-2 familyprotein(s) and induce apoptosis in the cell, wherein the cell is acancer cell.

In another aspect the disclosure provides methods for inducing apoptosisin a cell having a level of at least one of the BCL-2 family proteinmember greater than levels in a control cell, by administering to thecell an effective amount of a compound having structure A, or acombination thereof, or a pharmaceutically acceptable salt, hydrate,N-oxide, or solvate thereof, to reduce the level of BCL-2 familyprotein(s) and induce apoptosis in the cell, wherein the cell is acancer cell, wherein cancer is lung cancer, breast cancer, prostatecancer, or lymphomas.

In another aspect the disclosure provides methods for inducing apoptosisin a cell having a level of at least one of the BCL-2 family proteinmember greater than levels in a control cell, by administering to thecell an effective amount of a compound having structure A, or acombination thereof, or a pharmaceutically acceptable salt, hydrate,N-oxide, or solvate thereof, to reduce the level of BCL-2 familyprotein(s) and induce apoptosis in the cell, wherein the cell is a cellof the immune system.

In another aspect the disclosure provides methods for determining theeffectiveness of a therapeutic regimen including administration of acompound or stereoisomer thereof as described herein, or a combinationthereof, or a pharmaceutically acceptable salt, hydrate, N-oxide, orsolvate thereof, in a subject by comparing the level of a BCL-2 familyprotein in a cell of the subject prior to and during treatment with thecompound, or a pharmaceutically acceptable salt, hydrate, N-oxide, orsolvate thereof, wherein a decreased level of BCL-2 family protein isindicative of effectiveness of the therapy that utilizes the compound,or a pharmaceutically acceptable salt, hydrate, N-oxide, or solvatethereof.

In another aspect the disclosure provides methods for determining theeffectiveness of a therapeutic regimen including administration of acompound or stereoisomer thereof as described herein, or a combinationthereof, or a pharmaceutically acceptable salt, hydrate, N-oxide, orsolvate thereof, in a subject by comparing the level of a BCL-2 familyprotein in a cell of the subject prior to and during treatment with thecompound, or a pharmaceutically acceptable salt, hydrate, N-oxide, orsolvate thereof, wherein a decreased level of BCL-2 family protein isindicative of effectiveness of the therapy that utilizes the compound,or a pharmaceutically acceptable salt, hydrate, N-oxide, or solvatethereof, wherein the subject has cancer.

In another aspect the disclosure provides methods for determining theeffectiveness of a therapeutic regimen including administration of acompound or stereoisomer thereof as described herein, or a combinationthereof, or a pharmaceutically acceptable salt, hydrate, N-oxide, orsolvate thereof, in a subject by comparing the level of a BCL-2 familyprotein in a cell of the subject prior to and during treatment with thecompound, or a pharmaceutically acceptable salt, hydrate, N-oxide, orsolvate thereof, wherein a decreased level of BCL-2 family protein isindicative of effectiveness of the therapy that utilizes the compound,or a pharmaceutically acceptable salt, hydrate, N-oxide, or solvatethereof, wherein the subject has cancer, wherein cancer is lung cancer,breast cancer, prostate cancer, or lymphomas.

In another aspect the disclosure provides methods for determining theeffectiveness of a therapeutic regimen including administration of acompound or stereoisomer thereof as described herein, or a combinationthereof, or a pharmaceutically acceptable salt, hydrate, N-oxide, orsolvate thereof, in a subject by comparing the level of a BCL-2 familyprotein in a cell of the subject prior to and during treatment with thecompound, or a pharmaceutically acceptable salt, hydrate, N-oxide, orsolvate thereof, wherein a decreased level of BCL-2 family protein isindicative of effectiveness of the therapy that utilizes the compound,or a pharmaceutically acceptable salt, hydrate, N-oxide, or solvatethereof, wherein the subject has an autoimmune disorder.

In another aspect the disclosure provides methods for treating a diseaseor a disorder, by administering to a subject in need thereof atherapeutically effective amount of a compound or stereoisomer thereofas described herein, or a combination thereof, or a pharmaceuticallyacceptable salt, hydrate, N-oxide, or solvate thereof, thereby treatingthe disease or the disorder,

wherein the subject is afflicted with a condition wherein the conditionis lupus erythmatosus, psoriasis, psoriatic arthritis, lupus nephritis,rheumatoid arthritis, multiple sclerosis, ulcerative colitis, myastheniagravis, ITP, TTP, Grave's disease, Hashimoto's thyroiditis, Crohn'sdisease, autoimmune hemolytic anemias, insulin dependent diabetesmellitus, glomerulonephritis, rheumatic fever, osteoarthritis, goutyarthritis, dermatitis, bronchitis, rhinitis, asthma, Sjogrens' syndrome,meningitis, adrenoleukodystrophy, CNS vasculitis, mitochondrialmyopathies, Amyotrophic Lateral Sclerosis, Alzheimer's disease, or atumor.

In another aspect the disclosure provides methods for treating a diseaseor a disorder, by administering to a subject in need thereof atherapeutically effective amount of a compound or stereoisomer thereofas described herein, or a combination thereof, or a pharmaceuticallyacceptable salt, hydrate, N-oxide, or solvate thereof, thereby treatingthe disease or the disorder, wherein the subject is afflicted with acondition wherein the condition is lupus erythmatosus, psoriasis,psoriatic arthritis, lupus nephritis, rheumatoid arthritis, multiplesclerosis, ulcerative colitis, myasthenia gravis, ITP, TTP, Grave'sdisease, Hashimoto's thyroiditis, Crohn's disease, autoimmune hemolyticanemias, insulin dependent diabetes mellitus, glomerulonephritis,rheumatic fever, osteoarthritis, gouty arthritis, dermatitis,bronchitis, rhinitis, asthma, Sjogrens' syndrome, meningitis,adrenoleukodystrophy, CNS vasculitis, mitochondrial myopathies,Amyotrophic Lateral Sclerosis, Alzheimer's disease, or a tumor, whereinthe mitochondrial myopathy is MELAS syndrome, MERF syndrome, Leber'sdisease, Wernicke's encephalopathy, Rett syndrome, homocystinuria,hyperprolinemia, nonketotic hyperglycinemia, hydroxybutyricaminoaciduria, sulfite oxidase deficiency, or combined systems disease(B12 deficiency).

In another aspect the disclosure provides methods for determining theeffectiveness of a therapeutic regimen including administration of acompound or stereoisomer thereof as described herein, or a combinationthereof, or a pharmaceutically acceptable salt, hydrate, N-oxide, orsolvate thereof, in a subject by comparing the level of a BCL-2 familyprotein in a cell of the subject prior to and during treatment with thecompound, or a pharmaceutically acceptable salt, hydrate, N-oxide, orsolvate thereof, wherein a decreased level of BCL-2 family protein isindicative of effectiveness of the therapy that utilizes the compound,or a pharmaceutically acceptable salt, hydrate, N-oxide, or solvatethereof, wherein the subject has an autoimmune disorder, andadministering a selective serotonin reuptake inhibitor (SSRI).

In another aspect the disclosure provides methods for treatinginflammation in a subject, by administering to the subject in need ofsuch treatment an anti-inflammatory agent is gossypol, apogossypol,L-apogossypol, derivatives or stereoisomers of apogossypol, theaflavin,theaflavin-3′-gallate, theaflavanin, (−) gallocatechin-3-gallate (GCG),(−) epigallocatechin-3-gallate (EGCG), (−) catechin-3-gallate (CG), (−)epicatechin-3-gallate (ECG), or derivatives of purpurogallin, to treatthe inflammation thereby.

In another aspect the disclosure provides methods for treatinginflammation in a subject, by administering to the subject in need ofsuch treatment an anti-inflammatory agent is gossypol, apogossypol,L-apogossypol, derivatives or stereoisomers of apogossypol, theaflavin,theaflavin-3′-gallate, theaflavanin, (−) gallocatechin-3-gallate (GCG),(−) epigallocatechin-3-gallate (EGCG), (−) catechin-3-gallate (CG), (−)epicatechin-3-gallate (ECG), or derivatives of purpurogallin, to treatthe inflammation thereby, and administering a selective serotoninreuptake inhibitor (SSRI).

In another aspect the disclosure provides methods for treatinginflammation in a subject, by administering to the subject in need ofsuch treatment an anti-inflammatory agent is a stereoisomer ofapogossypol.

As mentioned herein, inflammation disorders may involve the activity ofapoptotic regulators. Thus, it is desirable to identify compounds thatmodulate the activity of apoptotic regulators, such as BCL-2 proteins.Such compounds are described herein. In some embodiments, the binding ofthese compounds prevents the interaction of anti-apoptotic BCL-2 familymembers with pro-apoptotic BCL-2 family members, and thereby reduces thebiological activity of anti-apoptotic BCL-2 family members. As a result,the compounds can be used to treat or prevent inflammatory disordersinvolving anti-apoptotic BCL-2 protein activity. In various embodiments,the compounds of interest comprise apogossypol, including (−)apogossypol substantially free of (+) apogossypol, as well as variousderivatives of apogossypol and other related compounds described herein.Such compounds can be administered to a patient with a highsusceptibility to developing a condition associated with inflammation,for example, lupus erythematosus, to reduce the likelihood that thepatient will develop such conditions.

As shown herein, apogossypol is more efficacious than gossypol, yet lesstoxic. The aldehydes in gossypol make it compound reactive, thuseffectively reducing the available concentrations of active drug andcausing toxicity. Apogossypol, a gossypol analog without the problematicaldehydes, retains full activity against anti-apoptotic BCL-2-familyproteins. Daily dosing studies, described in more detail in the Examplesportion of the application, show that mice tolerate doses of apogossypolabout 2-4-times higher than gossypol. Furthermore, the studies show thatapogossypol is superior to parent compound gossypol with respect totoxicology and efficacy.

In the general structure B shown herein, some specific R⁶, R⁸, R⁹ andR¹⁰ groups that may be used include, independently, hydrogen, —OH,—OCH₃, —CF₃, —CH₃, —OC₂H₅, —OC(O)CH₃, F, Cl, or Br. Some specific R⁷groups that may be used include, independently, hydrogen, —C₂H₅; -i-Pr,n-Pr, n-Bu, t-Bu, i-Bu, s-Bu, or cyclohexyl.

In some embodiments the compound of the general structure B shown hereinis apogossypol. The use of apogossypol for treating cancer is describedin PCT Publication No. WO 2005/009434, filed Jun. 25, 2005, which ishereby incorporated by reference in its entirety.

One specific compound of the disclosure described the general structureB shown herein has each of R⁶, R⁸, R⁹ as the acetate moiety (—OC(O)CH₃),has R⁷ as i-Pr, and R¹⁰ as —CH₃ (apogossypol hexacetate). This compoundcan also be used as pro-drug for oral administration of apogossypol. Inanother embodiment the compounds of the disclosure include compounds offormula B, where one of the R⁶ groups is a group other than hydrogen. Inone embodiment, the compound can be (−) apogossypol. In otherembodiments, the compound can be (−) apogossypol, (+) apogossypol,racemic apogossypol, S-apogossypol, R-apogossypol, or mixtures thereof.In another embodiment, the compound is substantially pure(−)apogossypol. In some embodiments, (−) apogossypol is at least 80percent of all macromolecular species in the composition, such as morethan about 85%, 90%, 95%, and 99%. For example, (−) apogossypol may bepurified to essential homogeneity, where the composition consistsessentially of solely (−) apogossypol. In various embodiments, thecompound is (−) apogossypol is substantially free of (+) apogossypol. Insome embodiments the compound of the general structure B shown herein iscompound XXI or XXII shown herein.

In one embodiment, the compound the general structure B shown hereincontains about 50% or more by weight of the (−) enantiomer ofapogossypol and about 50% or less by weight of (+) enantiomer ofapogossypol. In certain embodiments, the compound contains about 60% ormore by weight of the (−) enantiomer of apogossypol and about 40% orless by weight of (+) enantiomer of apogossypol. In some embodiments,the compound contains about 70% or more by weight of the (−) enantiomerof apogossypol and about 30% or less by weight of (+) enantiomer ofapogossypol. In some embodiments, the compound contains about 80% ormore by weight of the (−) enantiomer of apogossypol and about 20% orless by weight of (+) enantiomer of apogossypol. In some embodiments,the compound contains about 90% or more by weight of the (−) enantiomerof apogossypol and about 10% or less by weight of the (+) enantiomer ofapogossypol. In some embodiments, the compound contains about 95% ormore by weight of the (−) enantiomer of apogossypol and about 5% or lessby weight of (+) enantiomer of apogossypol. In some embodiments,apogossypol contains about 99% or more by weight of the (−) enantiomerof apogossypol and about 1% or less by weight of (+) enantiomer ofapogossypol.

The natural product Gossypol (1) is a potent inhibitor of Bcl-2,Bcl-X_(L) and Mcl-1, functioning as a BH3 mimic. (−) Gossypol iscurrently in phase clinical II trail, displaying single-agent antitumoractivity in patients with advanced malignancies. Given that Gossypol hastoxicity problems likely due to two reactive aldehyde groups, wedesigned Apogossypol (2), a compound that lacks these aldehydes, butretains activity against anti-apoptotic Bcl-2 family proteins in vitroand in cells. Recently, we also compared the efficacy and toxicity inmice of Gossypol and Apogossypol. Our preclinical in vivo data show thatApogossypol has superior efficacy and markedly reduced toxicity comparedto Gossypol. We also evaluated the single-dose pharmacokineticcharacteristics of Apogossypol in mice. Apogossypol displayed superiorblood concentrations over time compared to Gossypol, due to slowerclearance. These observations indicate that Apogossypol is a promisinglead compound for cancer therapy. Recently, we reported the separationand characterization of Apogossypol atropoisomers. These studiesrevealed that the racemic Apogossypol is as effective as its individualisomers. We further reported the synthesis and evaluation of 5,5′ ketonesubstituted Apogossypol derivatives and the best compound 3 (BI79D10)displayed improved in vitro and in vivo efficacy compared toApogossypol. However, compound 3 displayed mild GI toxicity during thecourse of transgenic mice studies while Apogossypol show no significantsign of toxicity which is likely due to relatively active ketone groupsin compound 3. In turn, we now place our attention on preparing andevaluating activities of novel 5,5′ substituted Apogossypol derivativeswhich replace reactive ketone groups with more druggable amide and alkylgroups at 5,5′ position.

Apogossypol is a promising inhibitor of Bcl-X_(L) and Bcl-2 withimproved in vivo efficacy and reduced toxicity compared to Gossypol.Molecular docking studies of Apogossypol into the BH3 binding groove inBcl-2 suggest that Apogossypol forms two hydrogen bonds with residuesArg 143 and Tyr 105 in Bcl-2 through 1 and 1′ hydroxyl group,respectively. Apogosypol also forms hydrogen bonds with Trp141 and Tyr199 in Bcl-2 through 6′ hydroxyl group on the right naphthalene ring.The isopropyl group on the left naphthalene ring inserts into the firsthydrophobic pocket (P1) in Bcl-2, while the isopropyl group on the rightnaphthalene ring inserts into the hydrophobic pocket (P2). Analysis ofthe predicted binding models indicates that while the overall corestructure of Apogossypol fits rather well into BH3 binding groove ofBcl-2, the two isopropyl groups do not apparently fully occupy thehydrophobic pockets P1 and P2. Therefore, a library of 5,5′ substitutedApogossypol derivatives that replace the isopropyl groups with suitablesubstituents was designed with the aim of deriving novel molecules thatcould occupy the hydrophobic pockets on Bcl-2 more efficiently.

A synthetic route was developed to install variety of amide groups at5,5′ position. Compound 1 (Gossypol) was treated with NaOH solution at90° C. to provide compound 2 (Apogossypol), which was readily methylatedby dimethyl sulfate in the presence of potassium carbonate to affordcompound 4. Reaction of compound 4 with TiCl₄ followed by dichloromethylmethyl ether at room temperature resulted in loss of isopropyl groupsand simultaneous bisformylation to give aldehyde compound 5. Thealdehyde groups of compound 5 were convert to carboxylic acid 6 by mildoxidation with sodium hypochlorite. The carboxylic acid 6 was thencoupled with a variety of commercially available amines in the presenceof 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide (EDCI) at roomtemperature to give compound 7. Subsequent demethylation of the compound7 using boron tribromide afforded compound 8. The synthesis of 5,5′alkyl substituted Apogossypol derivatives were outlined in FIGS. 12 and16-18. Compound 5 was treated with different Grignard or lithiumreagents to afford a secondary alcohol 9, which was oxidized to give thephenone 10 by pyridinium chlorochromate. Triethylsilane reduced phenone10 to alkyl compound 11 followed by subsequent demethylation using borontribromide to afford compound 12. Compounds 13 and 14, with hydrogenatom or carboxylic acid at 5,5′ positions, were synthesized to exploreif substitution at 5,5′ position is important for enhancing biologicalactivities. Compound 13 was synthesized by treating compound 4 withconcentrated sulfuric acid to lose isopropyl group. The resultingproduct and compound 6 was then treated individually with borontribromide to give compounds 13 and 14, respectively.

The synthesized 5,5′ substituted Apogossypol derivatives were firstscreened by one-dimensional ¹H nuclear magnetic resonance spectroscopy(1D-¹H NMR) binding assays against Bcl-X_(L). Active compounds in 1D-¹HNMR binding assays were then selected and evaluated in IsothermalTitration Calorimetry assays (ITC), cell viability assays andcompetitive fluorescence polarization assays (FPA). A group of compounds(8r, 8q, 8m) displayed high binding affinity for Bcl-X_(L) in theseassays. The most potent compound 8r induced significant chemical shiftchanges in active site methyl groups (region between −0.38 and 0.42 ppm)in the one-dimensional ¹H-NMR spectra of Bcl-X_(L) and also has an IC₅₀value of 0.76 μM in the FP displacement assays, which is 5 times moreeffective than Apogossypol. To confirm results of the NMR binding dataand the FP assays, we further evaluated the binding affinity of compound8r for Bcl-X_(L) using ITC assay. In agreement with NMR binding and FPAdata, compound 8r displayed potent binding affinity to Bcl-X_(L) with aK_(d) value of 0.11 μM, which is 15 times more potent than Apogossypol(K_(d)=1.7 μM) in the same assay. Consistent with NMR binding, FPA, andITC data, compound 8r displayed strong efficacy in inhibiting cellgrowth in PC3ML cells, which express high levels of Bcl-X_(L). The EC₅₀value of 8r is 1.7 μM, hence 6 fold more potent than Apogossypol(EC₅₀=10.4 μM). Compounds (8j-8s) displayed similar binding affinity as8r for Bcl-X_(L) in these assays with average IC₅₀ value of 2.8 μM.

Bcl-2 and Mcl-1 play critical roles in cell apoptosis and Bfl-1 has beenrecently suggested to be an important anti-apoptotic factor in largeB-cell lymphomas among Bcl-2 family proteins. Therefore we furtherevaluated the binding properties and specificity of selected Bcl-X_(L)active 5,5′ substituted Apogossypol derivatives against Bcl-2, Mcl-1 andBfl-1 using FP assays. Compound 8r displayed strong binding affinity forBcl-2, Mcl-1 and Bfl-1. It inhibited Bcl-2, Mcl-1 and Bfl-1 with IC₅₀values of 0.32, 0.28 and 0.73 μM, respectively, which are approximately10 times more potent than Apogossypol in similar FP assays. Compound 8rwas further evaluated against H460, H1299 and BP3 cell lines, whichexpress high levels of Bcl-2, Mcl-1 and Bfl-1, respectively. Consistentwith FPA data, compound 8r displayed significant efficacy in inhibitingcell growth in H460 and BP3 cells with IC₅₀ value of 0.33 μM and 0.66μM, respectively, which are approximately 7-10 times more potent thanApogossypol. Molecular docking studies of compound 8r demonstrated that2-phenylpropyl groups at 5,5′ positions inserted deeper into hydrophobicpockets (P1 and P2) in Bcl-2, hence occupying these regions moreefficiently compared to isopropyl groups of Apogossypol. In addition,the carbonyl group on the right naphthalene ring also formed anadditional hydrogen bond with residue Tyr199. However, compound 8rdisplayed similar cell activity in H1299 cell line compared toApogossypol which is probably because different cell lines havedifferent sensitivities for compound 8r. Other 5,5′ substitutedApogossypol derivatives, such as 12e, 8n, 8p, 8q, 8k also displayedstrong pan-active inhibitory properties against Bcl-2, Mcl-1 and Bfl-1.The most potent compound 8q bound to Bcl-2, Mcl-1 and Bfl-1 with IC₅₀value of 0.67, 0.59 and 1.3 μM, respectively, in FP assays. In agreementwith FPA, it show potent cell inhibition activity for H460, H1299 andBP3 cell lines with IC₅₀ value of 0.40, 0.36 and 0.20 μM, respectively.

Analysis of structure-activity relationship (SAR) of synthesized 5,5′substituted Apogossypol derivatives reveals that substitution at 5,5′position are important for achieving stronger binding affinity to Bcl-2family proteins. Accordingly compounds 13 and 14 with hydrogen atoms orcarboxylic acid groups on 5,5′ positions, displayed weak or noinhibition in all assays. Analysis of SAR of 5,5′ amide substitutedApogossypol derivatives furthter indicates that longer and flexiblehydrophobic groups show better efficacy than small, short and rigidhydrophobic groups. Replacement of small methylcyclopropane (8l) orshort cyclopentyl (8b) group by longer methylcyclohexyl group (8m)significantly increased cell inhibition potency. Also, compounds (8n-8s)having phenethyl groups at 5,5′ positions displayed potent cell activityin the H460 and PC3ML cell lines with average EC₅₀ values of 0.64 μM and2.6 μM, respectively while compounds (8a-8e) having phenyl groupdisplayed relatively weak cell activity with average EC₅₀ values of 8.6μM and 11.3 μM, respectively. Based on our modeling prediction, this islikely because longer and flexible groups could insert deeper into theP1 and P2 pockets. We also explored the SAR of the 5,5′ alkylsubstituted Apogossypol derivatives. In general, longer hydrophobicgroups also show improved potency. Compounds 12a and 12b with isobutyland isopentyl groups displayed improved activity compared to Apogossypolwith isopropyl groups. Again, compound 12e with phenethyl group are moreactive than compound 12d with benzyl group.

The H460 cell line has been studied by several groups. A pan-Bcl-2family inhibitor, GX15-070, was tested in H460 cell line with an IC₅₀value of 3.85 μM. BP3 is human diffuse large B-cell lymphoma (DLBCL)cell line overexpressing Bfl-1. The mRNA ratio of Bfl-1, Bcl-X_(L) andMcl-1 is approximately 10:3:1. We determined that BP3 cell overexpressedhigh level Bfl-1 and Mcl-1 by Western blot analysis as shown below inTable 1.

TABLE 1 Mcl-1 Bcl-2 Bcl-xl Bfl-1 BP3 +++ No + +++ RS4; 11 No ++++ + No4-point rating scale for western data: ++++: Very high level +++: Highlevel ++: Medium level +: Low No: Not Detectable

The potent Bcl-X_(L) and Bcl-2 antagonist ABT-737 displayed no cellactivity against BP3 cell lines because ABT737 is not effective againstMcl-1 and Bfl-1.

Hence, we next evaluated the ability of 5,5′ substituted Apogossypolderivatives to induce apoptosis of the human lymphoma RS11846 cell line,which expresses high levels of Bcl-2 and Bcl-X_(L). For these assays, weused Annexin V-FITC and propidium iodide (PI) double staining, followedby flow-cytometry analysis. Most of synthesized Apogossypol derivativeseffectively induced apoptosis of the RS11846 cell line in adose-dependent manner. In particular, compounds 8q, 8r and 8n areeffective with EC₅₀ values ranging from 3.0 to 5.8 μM, which isconsistent with previous results in human cancer PC3ML and H460 celllines. Again, the negative control compounds 13 and 14 induced weak orno apoptosis of the RS11846 cell line.

We also explored whether 5,5′ substituted Apogossypol derivatives hadcytotoxicity against Bax/Bak double knockout (DKO) mouse embryonicfibroblast cells (MEF) in which antiapoptotic Bcl-2 family proteins lacka cytoprotective phenotype. Some potent pan-active Bcl-2 compounds (8m,8q, 8r, 8k, 8p, 12e) displayed slightly cytotoxicity in Bax/Bak doubleknockout mouse embryonic fibroblast cells (MEF/DKO) by killing 20-35% ofthem at 10 μM using FITC-Annexin V/PI assays, implying that thosecompounds displayed some off-target effects. However, those compoundsdisplayed reduced off-target effects than Gossypol which displayed verysimilar cytotoxicity in MEF and MEF/DKO cells at 10 μM. In comparison,Apogossypol had reduced off-target effect but displayed weaker abilityto induce apoptosis of the MEF cells compared to 5,5′ amide substitutedApogossypol derivatives.

Apogossypol is a polyphenol scaffold with 6 hydroxyl groups on thenaphthalene ring which can be oxidized to quinones. We previouslystabilized Apogossypol by cocrystallizing it with ascorbic acid.Apogossypol can also be stabilized by introducing electron withdrawinggroups, such as carbonyl groups on the naphthalene rings because thesewill decrease the electron density on the naphthalene ring andsubsequently slow down oxidation rate and other side reactions. Thechemical stability of solid compounds (8m, 8q, 8r, 8k, 8p, 12e) wasevaluated at room temperature. The stability of compound was monitoredusing a combination of HPLC and LCMS. Overall, 5,5′ amide substitutedApogossypol derivatives show superior chemical stability compared toApogossypol. In particular, 8r and 8q were 10% degraded after 60 days atroom temperature while Apogossypol is almost 80% decomposed under samecondition in the absence of ascorbic acid. Compound 12e having phenethylgroup at 5,5′ position are also less stable than amide compounds due tolack of electron withdrawing groups.

To test the pharmacological properties of 5,5′ substituted Apogossypolderivatives, we determined their in vitro plasma stability, microsomalstability, and cell membrane permeability. From these studies, we couldconclude that our synthesized compounds displayed superior plasma andmicrosomal stability than Apogossypol. Compounds 8r and 8m degraded 4%and 11%, respectively, after 1 hour incubation in rat plasma whileApogossypol degraded 47% under same condition. In addition, compounds 8rand 8m degraded 24% and 10%, respectively, after 1 hour incubation inrat microsomal preparations while Apogossypol degraded 36% under samecondition. Compounds 8r and 8m also showed similar or improved cellmembrane permeability compared to Apogossypol.

Hence, using a combination of 1D ¹H-NMR binding assays, FP assays, ITCassays, cytotoxicity assays and preliminary in vitro ADME data,compounds such as 8r and 8q were selected for further in vivo studiesusing B6Bcl-2 transgenic mice. B-cells of the B6Bcl-2 transgenic miceoverexpress human Bcl-2 and accumulate in the spleen of mice. The spleenweight is used as an end-point for assessing in vivo activity as wedetermined that the spleen weight is highly consistent in age- andsex-matched Bcl-2-transgenic mice and variability was within ±2% amongcontrol B6Bcl2 mice. We first screened the in vivo activities ofcompounds such as 8r and 8q side by side with Apogossypol and Gossypolin a single Bcl-2 transgenic mouse with a single intraperitoneal (ip)injection at 72 μmol/kg. In agreement with all in vitro data, tested5,5′ amide substituted Apogossypol derivatives displayed superior invivo activity compared to Apogossypol and Gossypol. In particular,compounds (8r, 8k and 8p) induced more than 40% spleen weight reduction.Since the maximum spleen shrinkage would be no more than 50% in thisexperimental model, these compounds induced near maximal (85-95%)biological activity while Apogossypol and Gossypol induced 40% ofmaximum reduction in spleen weight at same dose. Again, the negativecontrol, compound 13 displayed no activity in transgenic mice model, asexpected. Overall tested 5,5′ alkyl substituted Apogossypol derivatives(12c and 12e) displayed lower in vivo activity compared to 5,5′ amidesubstituted Apogossypol derivatives. However, the 5,5′ alkyl substitutedApogossypol show no significant signs of toxicity at 72 μmol/kg and evenat 120 μmol/kg while 5,5′ amide substituted Apogossypol show toxicitysigns at 72 μmol/kg as shown below in Table 2.

TABLE 2 2 1 13 12e 8k 8m 8p 8q 8r 12c Effica PR PR NR PR CR PR CR PR CRPR Tox 0 2+ 0 0 4+ 4+ 4+ 4+ 3+ 1+ Toxicity Rating Scale: 4+ (lethal),3+: severe, 2+: moderate, 1+: mild, 0: No toxicity

The mice treated with compound 8r had more apparent signs of GI toxicityat 72 μmol/kg (50 mg/kg). In order to balance the toxicity and efficacyof compound 8r, we next explored the maximum tolerated dose (MTD) of 8rusing a group of five mice. Mice were treated with a single dose of 100,75, 50, 25 and 12.5 mg/kg (ip) and observed for a period of 14 daysmonitoring morbidity (body weight loss) and mortality. All mice werealive after 14 days and the maximum weight loss was observed at thefifth day which underwent 80-100% recovery after 14 days. The mice dosedat 25 and 12.5 mg/kg showed no weight loss while the mice dosed at 50mg/kg displayed around 13% weight loss. Therefore the MTD of compound 8ris likely between 25 mg to 50 mg/kg, approximately. We next evaluatedthe in vivo activity and toxicity of the compound 8r in groups of sixmice each at dose of 42 mg/kg (60 μmol/kg). Consistent with the singlemouse experiment, compound 8r treatment of these mice resulted in asignificant (˜70%) reduction of spleen weight (P<0.0001) compared to thecontrol group of six mice. All mice tolerated the treatment well andmild signs of GI toxicity were observed. The average weight loss of micewas 7.8% during the course of this study with 8r.

In summary, a library of 5,5′ substituted Apogossypol derivatives wassynthesized and evaluated in a variety of in vitro and in vivo assays.The most potent compound, 8r, was found to bind to Bcl-X_(L), Bcl-2,Mcl-1 and Bfl-1 with IC₅₀ values of 760 nM, 320 nM, 280 nM and 730 nM,respectively. The compound also potently inhibited growth in cellcultures of the PC3ML, H460, H1299 and BP3 cancer cell lines, whichexpress Bcl-X_(L), Bcl-2, Mcl-1 and Bfl-1, respectively, with EC₅₀values in the submicromolar to nanomolar range. Compound 8r effectivelyinduced apoptosis of the RS11846 human lymphoma cell line in adose-dependent manner and show little cytotoxicity against Bax/Bakdouble knockout mouse embryonic fibroblast cells in which antiapoptoticBcl-2 family proteins lack a cytoprotective phenotype, implying thatcompound 8r has little off-target effects. Finally, compound 8r showedfavorable chemical stability, in vitro ADME properties and superior invivo efficacy compared to Apogossypol in Bcl-2 transgenic mice in whichBcl-2 is overexpressed in B-cells. Considering the critical roles ofanti-apoptotic Bcl-2 family proteins in tumorgenesis, chemoresistance,and the potent inhibitory activity of 8r against anti-apoptotic Bcl-2family proteins, compound 8r res a viable drug candidate for thedevelopment of novel apoptosis-based cancer therapies.

TABLE 3 EVALUATION OF 5, 5′ SUBSTITUTED APOGOSSYPOL DERIVATIVES USING ACOMBINATION OF 1D ¹H-NMR BINDING ASSAYS AND CELL VIABILITY ASSAYS

EC50 (PM) Compound R = 1D-¹H NMRa* RS11846c* H1299b* H460b* PC3MLb*BP3c* Gossoypol (1) +++ 4.2 6.0 3.0 3.1 1.42 Apogossypol (2)

++ 5.0 3.4 3.5 10.4 4.7 14 COOH − >30 >30 >30 >30 >30 8a

+ 15.1 8.0 3.5 15.1 ND 8b

+ 13.7 8.0 8.5 12.2 ND 8c

+ 10.8 17.0 10.1 8.5 ND 8d

+ 4.7 5.0 4.1 8.3 ND 8e

+ 12.6 28.7 16.7 12.2 ND 8f

+++ 4.2 ND 1.5 ND ND 8g

+ 9.3 3.6 4.6 13.7 ND 8h

+++ 4.8 3.1 2.9 10.2 ND 8i

+++ 7.3 5.2 3.3 8.3 ND 8j

++ 3.0 3.6 1.4 0.7 1.5 8k

+++ 5.5 3.2 0.50 5.0 1.5 8l

+ >10 ND ND ND ND 8m

+++ 4.9 4.8 0.41 3.7 0.90 8n

++ 3.1 3.6 0.55 3.9 0.72 8o

+ 5.6 ND 0.70 ND ND 8p

+++ 4.6 7.8 0.99 3.2 0.41 8q

+++ 3.0 0.36 0.40 1.7 0.2 8r

++ 5.8 3.2 0.33 1.7 0.66 8s

++ 4.1 7.1 0.94 3.0 1.1 8t

++ 5.1 ND 1.3 ND 0.70 a*4-point-rating scale: +++, Very Active; ++,Active; +, Mild; −, Weak b*Compounds against cell line using ATP-LITEassay c*Compounds against cell line using Annexin V-FITC and propidiumiodide assay d*ND: not determined

TABLE 4 EVALUATION OF 5, 5′ SUBSTITUTED APOGOSSYPOL DERIVATIVES USING ACOMBINATION OF 1D ¹H-NMR BINDING ASSAYS AND CELL VIABILITY ASSAYS

EC₅₀ (μM) Compound R = 1D-¹H NMRa* RS11846c* H1299b* H460b* PC3MLb*BP3c* Apogossypol (2)

++ 5.0 3.4 3.5 10.4 4.7 13 H − 24.5 13.4 >10 >10 NDd* 12a

+ 8.4 1.2 3.2 6.7 ND 12b

+ 4.8 1.8 1.1 5.2 ND 12c

+ 4.5 10.9 1.8 11.2 ND 12d

+ 3.2 1.28 1.2 8.3 ND 12e

+ 9.8 0.58 0.92 2.4 4.14 a*4-point-rating scale: +++: Very Active; ++:Active; +: Mild; −: Weak b ^(*)Compounds against cell line usingATP-LITE assay c *Compounds against cell line using Annexin V-FITC andpropidium iodide assay d*ND: not determined

TABLE 5 CROSS-ACTIVITY OF SELECTED 5,5′ SUBSTITUTED APOGOSSYPOLDERIVATIVES AGAINST BCL-X_(L), BCL-2, MCL-1 and BFL-1 K_(d) (μM)Compound IC₅₀ (μM) FPA ITC IC₅₀ Bcl-X_(L) Bcl-2 Mcl-1 Bfl-1 Bcl-X_(L)Apogossypol (2) 3.7 4.3 2.6 >10 1.7  12e 3.5 0.48 0.83 5.0 0.41 8m 1.10.71 0.78 2.0 0.85 8n 0.80 0.15 0.30 0.55 ND 8p 6.3 4.4 3.2  ND^(a)* ND8q 0.93 0.67 0.59 1.3 0.12 8j 0.8 0.70 1.1 ND ND 8k 0.27 0.49 0.23 0.400.11 8r 0.76 0.32 0.28 0.73 0.11 8s 0.85 0.70 0.35 0.67 ND ND^(a)* = Notdetermined

TABLE 6 PLASMA STABILITY, MICROSOMAL STABILITY, AND CELL PERMEABILITY OFSELECTED 5,5′ SUBSTITUTED APOGOSSYPOL DERIVATIVES Plasma stabilityMicrosomal Cell Compound (T = 1 h) Stability (T = 1 h) PermeabilityApogossypol (1) 53% 64% −7.16 12e 80% 89% −6.61 12c 81% 75% −6.27 8n 63%60% −6.49 8m 89% 90% −6.67 8p ND^(a)* 90% −7.71 8q 94% 87% −8.15 8r 96%76% −7.51 8k 94% 71% −7.92 ND^(a)*= Not determined

Binding of the compounds disclosed herein to anti-apoptotic BCL-2proteins can induce apoptosis and thereby treat inflammation and/orinflammatory disorders. In some embodiments, the compounds disclosedherein can bind to anti-apoptotic BCL-2 family proteins such as, forexample, BCL-2 or BCL-X_(L). This binding can inhibit binding of theanti-apoptotic BCL-2 family members to pro-apoptotic BCL-2 familymembers. In various embodiments, binding of the compounds disclosedherein can reduce the formation of complexes between anti-apoptoticBCL-2 proteins and the BH3 domain of pro-apoptotic BCL-2 family members.

Guided by a combination of nuclear magnetic resonance (NMR) bindingassays and computational docking studies, a series of 5,5′ substitutedApogossypol derivatives were synthesized as potent pan-active inhibitorsof anti-apoptotic Bcl-2 family proteins. One of the most potentcompound, 8r, inhibits the binding of BH3 peptides to Bcl-X_(L), Bcl-2,Mcl-1 and Bfl-1 with IC₅₀ values of 0.76 μM, 0.32 μM, 0.28 μM and 0.73μM, respectively. This compound also potently inhibits cell growth inthe H460 human lung cancer and BP3 human B-cell lymphoma cell lines withEC₅₀ values of 0.33 μM and 0.66 μM, respectively. Compound 8reffectively induces apoptosis of the RS11846 human lymphoma cell line ina dose-dependent manner and shows little cytotoxicity againstbax^(−/−)bak^(−/−) cells in which antiapoptotic Bcl-2 family proteinslack a cytoprotective phenotype, implying that compound 8r has littleoff-target effect. Compound 8r also displays in vivo efficacy intransgenic mice in which Bcl-2 is overexpressed in splenic B-cells.Together with its improved chemical, plasma and microsomal stabilityrelative to Apogossypol, Compound 8r res a novel apoptosis-based therapyfor cancer.

According to other embodiments, the disclosure provides a method fortreating a disease or disorder. The method can include administering toa subject in need of such treatment, an effective amount of any hereindescribed compound, or pharmaceutically acceptable salts, hydrates, orsolvates thereof. Non-limiting examples of the diseases or disordersthat can be treated are cancer and autoimmune diseases.

According to another embodiment, the disclosure provides a method fortreating cancer. The method comprises administering to a subject in needthereof a therapeutically effective amount of any abo herein describedcompound, or pharmaceutically acceptable salts, hydrates, or solvatesthereof. Any herein described compound may be used for treating any typeof cancer. In some aspects, the kinds of cancer that may be treatedinclude lung cancer, breast cancer, prostate cancer, as well as avariety of lymphomas.

According to another embodiment, any of the disclosed compound can beused for the manufacture of a medicament for the treatment of apathological condition or symptom in a mammal, such as a human. Themedicament can be directed to the treatment of cancer, within thelimitations described herein.

According to another embodiment, the disclosure provides pharmaceuticalcompositions. The pharmaceutical compositions may comprise any of thedisclosed compounds, or pharmaceutically acceptable salts, hydrates, orsolvates thereof, and a pharmaceutically acceptable diluent or carrier.The pharmaceutical compositions can be used to treat cancer. Thepharmaceutical compositions can further optionally include one or moreadditional therapeutic anti-cancer agents, including, but not limitedto, such agents as (1) alkaloids, including, microtubule inhibitors(e.g., Vincristine, Vinblastine, and Vindesine, etc.), microtubulestabilizers (e.g., Paclitaxel [Taxol], and Docetaxel, Taxotere, etc.),and chromatin function inhibitors, including, topoisomerase inhibitors,such as, epipodophyllotoxins (e.g., Etoposide [VP-16], and Teniposide[VM-26], etc.), and agents that target topoisomerase I (e.g.,Camptothecin and Isirinotecan [CPT-11], etc.); (2) covalent DNA-bindingagents [alkylating agents], including, nitrogen mustards (e.g.,Mechlorethamine, Chlorambucil, Cyclophosphamide, Ifosphamide, andBusulfan [Myleran], etc.), nitrosoureas (e.g., Carmustine, Lomustine,and Semustine, etc.), and other alkylating agents (e.g., Dacarbazine,Hydroxymethylmelamine, Thiotepa, and Mitocycin, etc.); (3) noncovalentDNA-binding agents [antitumor antibiotics], including, nucleic acidinhibitors (e.g., Dactinomycin [Actinomycin D], etc.), anthracyclines(e.g., Daunorubicin [Daunomycin, and Cerubidine], Doxorubicin[Adriamycin], and Idarubicin [Idamycin], etc.), anthracenediones (e.g.,anthracycline analogues, such as, [Mitoxantrone], etc.), bleomycins(Blenoxane), etc., and plicamycin (Mithramycin), etc.; (4)antimetabolites, including, antifolates (e.g., Methotrexate, Folex, andMexate, etc.), purine antimetabolites (e.g., 6-Mercaptopurine [6-MP,Purinethol], 6-Thioguanine [6-TG], Azathioprine, Acyclovir, Ganciclovir,Chlorodeoxyadenosine, 2-Chlorodeoxyadenosine [CdA], and2′-Deoxycoformycin [Pentostatin], etc.), pyrimidine antagonists (e.g.,fluoropyrimidines [e.g., 5-fluorouracil (Adrucil), 5-fluorodeoxyuridine(FdUrd) (Floxuridine)] etc.), and cytosine arabinosides (e.g., Cytosar[ara-C] and Fludarabine, etc.); (5) enzymes, including, L-asparaginase,and hydroxyurea, etc.; (6) hormones, including, glucocorticoids, suchas, antiestrogens (e.g., Tamoxifen, etc.), nonsteroidal antiandrogens(e.g., Flutamide, etc.), and aromatase inhibitors (e.g., anastrozole[Arimidex], etc.); (7) platinum compounds (e.g., Cisplatin andCarboplatin, etc.); (8) monoclonal antibodies conjugated with anticancerdrugs, toxins, and/or radionuclides, etc.; (9) biological responsemodifiers (e.g., interferons [e.g., IFN-.alpha., etc.] and interleukins[e.g., IL-2, etc.], etc.); (10) adoptive immunotherapy; (11)hematopoietic growth factors; (12) agents that induce tumor celldifferentiation (e.g., all-trans-retinoic acid, etc.); (13) gene therapyagents; 14) antisense therapy agents; (15) tumor vaccines; (16) agentsdirected against tumor metastases (e.g., Batimistat, etc.); (17)inhibitors of angiogenesis, and (18) selective serotonin reuptakeinhibitors (SSRI's).

Reactive, but non-limiting examples of suitable SSRIs that may be usedinclude sertraline (e.g., sertraline hydrochloride, marketed under thetrademark “Zoloft®” by Pfizer, Inc.) or sertraline metabolite,fluvoxamine (e.g., fluvoxamine melate, marketed under the trademark“Luvox®” by Solvay Pharmaceuticals, Inc.), paroxetine (e.g., paroxetinehydrochloride, marketed under the trademark “Paxil®” by SmithKlineBeecham Pharmaceuticals, Inc.), fluoxetine (e.g., fluoxetinehydrochloride, marketed under the trademarks “Prozac®” or “Sarafem®” byEli Lilly and Company) and citalopram (e.g., citalopram hydrobromide,marketed under the trademark “Celexa®” by Forest Laboratories,Parke-Davis, Inc.), and metabolites thereof. Additional examples includevenlafaxine (e.g., venlafaxine hydrochloride marketed under thetrademark “Effexor®” by Wyeth-Ayerst Laboratories), mirtazapine (e.g.,marketed under the trademark Remeron® by Organon, Inc.), buspirone(e.g., buspirone hydrochloride marketed under the trademark “Buspar®” byBristol-Myers Squibb), trazodone (e.g., trazodone hydrochloride marketedunder the trademark “Desyrel®” by Bristol-Myers Squibb and Apothecon),nefazadone (e.g., nefazodone hydrochloride marketed under the trademark“Serzon®” by Bristol-Myers Squibb), clomipramine (e.g., clomipraminehydrochloride marketed under the trademark “Anafranil®” by Novopharm,LTD, Ciba, and Taro Pharmaceuticals), imipramine (e.g., imipraminehydrochloride marketed under the trademark Tofranil® by Glaxo-Welcome,Inc.), nortriptyline (e.g., Nortriptyline hydrochloride marketed underthe trademark “Nortrinel®” by Lundbeck), mianserine (e.g., marketedunder the trademark “Tolvon®” by Organon, Inc.), duloxetine (e.g.,duloxetine hydrochloride marketed by Eli Lilly and Company), dapoxetine(e.g., dapoxetine hydrochloride marketed by ALZA Corporation),litoxetine (e.g., litoxetine hydrochloride marketed by SynthelaboRecherche (L.E.R.S.), Bagneux, France.), femoxetine, lofepramine (e.g.,marketed under the trademark “Gamonil®” by MERCK & Co., Inc.),tomoxetine (e.g., marketed by Eli Lilly and Company). The disclosureencompasses SSRIs that are currently used, or those later discovered orformulated. SSRIs, including those listed herein, may be administeredorally in an amount between about 2 mg and about 2,500 mg daily.

In the broad sense, any cancer or tumor (e.g. hematologic and solidtumors) may be treated according to embodiments of the disclosure.Exemplary cancers that may be treated according to embodiments of thedisclosure include, but are not limited to, head and neck cancer, braincancer (e.g. glioblastoma multifoma) breast cancer, colorectal cancer,esophageal cancer, gastric cancer, hepatic cancer, bladder cancer,cervical cancer, endometrial cancer, lung cancer (non-small cell),ovarian cancer and other gynological cancers (e.g. tumors of the uterusand cervix), pancreatic cancer, prostate cancer, renal cancer,choriocarcinoma (lung cancer), skin cancer (e.g. melanoma, basal cellcarcinoma), hairy cell leukemia, chronic lymphotic leukemia, acutelymphocytic leukemia (breast & bladder), acute myelogenous leukemia,meningeal leukemia, chronic myelogenous leukemia, and erythroleukemia.More commonly, the cancers treated include leukemia and B-cell cancers(e.g. lymphoma, multiple myeloma, and MDS.

Non-limiting examples of autoimmune diseases that can be treated usingany herein described compound and methods of the disclosure includerheumatoid arthritis, psoriatic arthritis, juvenile idiopathicarthritis, multiple sclerosis, systemic lupus erythematosus, myastheniagravis, juvenile onset diabetes, glomerulonephritis, autoimmunethyroiditis, Behcet's disease, Crohn's disease, ulcerative colitis,bullous pemphigoid, sarcoidosis, psoriasis, ichthyosis, Gravesopthalmopathy, psoriasis, psoriasis inflammatory bowel disease, andasthma.

As discussed in more detail-herein, some embodiments also providemethods for treating and/or prevention various inflammatory disorders,diseases and conditions. Such inflammatory disorders, diseases andconditions include, without limitation, systemic autoimmune diseasessuch as, for example, lupus erythematosus, rheumatoid arthritis,multiple sclerosis, and psoriasis; and organ specific autoimmunediseases such as, for example, ulcerative colitis, myasthenia gravis,Grave's disease, Hashimoto's thyroiditis, Crohn's disease, lupusnephritis, autoimmune hemolytic anemias, immune thrombocytopenic purpura(ITP), thrombotic thrombocytopenic purpura (TTP), insulin dependentdiabetes mellitus, glomerulonephritis, and rheumatic fever. Otherinflammatory diseases that may be treated in accordance with thisdisclosure include, without limitation, other inflammatory arthriticconditions such as psoriatic arthritis, osteoarthritis and goutyarthritis, as well as other inflammatory conditions such asconjunctivitis, dermatitis, bronchitis, rhinitis etc., brought about byinjury, allergies, infections, microorganisms, trauma, or physical orchemical agents. The treatment of inflammatory aspects of asthma,Sjogrens' syndrome, meningitis, adrenoleukodystrophy, CNS vasculitis,mitochondrial myopathies, Amyotrophic Lateral Sclerosis, Alzheimer'sdisease, or tumors is also contemplated as part of this disclosure.Examples of mitochondrial myopathies include MELAS syndrome, MERFsyndrome, Leber's disease, Wernicke's encephalopathy, Rett syndrome,homocystinuria, hyperprolinemia, nonketotic hyperglycinemia,hydroxybutyric aminoaciduria, sulfite oxidase deficiency, and combinedsystems disease (B12 deficiency). In association with such preventionand/or treatment, articles of manufacture, compositions, methods of useand medical treatments by the compounds described herein are alsoprovided.

In some cases, it may be appropriate to administer any herein describedcompound as a salt. Examples of pharmaceutically acceptable saltsinclude organic acid addition salts formed with acids which form aphysiological acceptable anion, for example, tosylate, methanesulfonate,acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate,ketoglutarate, and glycerophosphate. Suitable inorganic salts may alsobe formed, including hydrochloride, sulfate, nitrate, bicarbonate, andcarbonate salts. Pharmaceutically acceptable salts may be obtained usingstandard procedures well known in the art, for example by reacting anyherein described compound with a suitable base affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

Any tablets, troches, pills, capsules, and the like, which incorporateany herein described compound, may also contain binders such as gumtragacanth, acacia, corn starch or gelatin; excipients such as dicalciumphosphate; a disintegrating agent such as corn starch, potato starch,alginic acid and the like; a lubricant such as magnesium stearate; and asweetening agent such as sucrose, fructose, lactose or aspartame or aflavoring agent such as peppermint, oil of wintergreen, or cherryflavoring may be added. When there is a unit dosage form of any hereindescribed compound, it may contain, in addition to materials of theherein type, a liquid carrier, such as a vegetable oil or a polyethyleneglycol. Various other materials may be as coatings or to otherwisemodify the physical form of a solid unit dosage form. For instance,tablets, pills, or capsules may be coated with gelatin, wax, shellac orsugar and the like. A syrup or elixir may contain the active compound,sucrose or fructose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Anymaterial used in preparing any unit dosage form should bepharmaceutically acceptable and substantially non-toxic in the amountsemployed. In addition, any herein described compound may be incorporatedinto sustained-release preparations and devices.

Any herein described compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of any hereindescribed compound may be prepared in water, optionally mixed with anontoxic surfactant. Dispersions may also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationsmay contain a preservative to prevent the growth of microorganisms.

Sterile injectable solutions can be prepared by incorporating any hereindescribed compound of in the sufficient therapeutic amount in theappropriate solvent with various of the other ingredients enumeratedherein, as required, followed by filter sterilization. In the case ofsterile powders for the preparation of sterile injectable solutions, themethods of preparation are vacuum drying and the freeze dryingtechniques, which yield a powder of the active ingredient plus anyadditional desired ingredient in the previously sterile-filteredsolutions.

For topical administration, any herein described compound may be appliedin pure form, i.e., when it is a liquid. However, it will generally bedesirable to administer it to the skin as compositions or formulations,in combination with a dermatologically acceptable carrier, which may bea solid or a liquid. Useful solid carriers include finely divided solidssuch as talc, clay, microcrystalline cellulose, silica, alumina and thelike. Useful liquid carriers include water, alcohols or glycols orwater-alcohol/glycol blends, in which the compounds can be dissolved ordispersed at effective levels, optionally with the aid of non-toxicsurfactants. Adjuvants and additional antimicrobial agents can be addedto optimize the properties for a given use.

The resultant liquid compositions can be applied from absorbent pads,used to impregnate bandages and other dressings, or sprayed onto theaffected area using pump-type or aerosol sprayers. Thickeners such assynthetic polymers, fatty acids, fatty acid salts and esters, fattyalcohols, modified celluloses or modified mineral materials can also beemployed with liquid carriers to form spreadable pastes, gels,ointments, soaps, and the like, for application directly to the skin ofthe user, as known to those having ordinary skill in the art.

The disclosure also provides a pharmaceutical composition of thecompounds described herein, or a pharmaceutically acceptable saltthereof, in combination with a pharmaceutically acceptable diluent orcarrier. Further, the disclosure provides the use of compounds disclosedherein in combination with other known anti-inflammatory compounds.

In various embodiments, the disclosure provides a method for treatinginflammatory disease and/or a condition associated with inflammation byadministering to a mammal in need of such therapy, an effective amountof the compounds described herein, the compounds described herein incombination with an additional anti-inflammatory compound or apharmaceutically acceptable salt thereof. In other embodiments, methodsfor the prevention of inflammatory disease and/or a condition associatedwith inflammation or a method for reducing the likelihood that a patientwill develop such inflammation is provided. The methods can includeadministering to a mammal in need of such therapy, an effective amountof the compounds described herein or a pharmaceutically acceptable saltthereof.

There are also provided methods for treating a mammalian subject,particularly a human, suspected of having, or being prone to a diseaseor condition involving inflammation, by administering to a mammaliansubject in need thereof a therapeutically effective amount of a compoundby at least one of the compounds of the general structure B shownherein, a single enantiomer of a compound of the general structure B, amixture of the (+) enantiomer and the (−) enantiomer, a mixture of about90% or more by weight of the (−) enantiomer and about 10% or less byweight of the (+) enantiomer, an individual diastereomer of a compoundof the general structure B, a mixture of diastereomers, or apharmaceutically acceptable salt, solvate, or prodrug thereof, so as toaffect treat or prevent inflammation. In some embodiments, the compoundis apogossypol.

In some embodiments, the methods for treating inflammation or preventinginflammation include administration of an effective amount of anothertherapeutic agent useful for treating or preventing the diseases ordisorders disclosed herein. In some embodiments, the time in which thetherapeutic effect of the other therapeutic agent is exerted overlapswith the time in which the therapeutic effect of the apogossypol orderivative is exerted.

In some embodiments, the other therapeutic agent is an anti-inflammatoryagent. Examples of anti-inflammatory agents suitable for use accordingto some embodiments disclosed herein include, but are not limited to,steroids (e.g., cortisol, cortisone, fludrocortisone, prednisone,methylprednisolone, 6-methylprednisone, triamcinolone, betamethasone ordexamethasone), nonsteroidal anti-inflammatory drugs (NSAIDS (e.g.,aspirin, acetaminophen, tolmetin, salicylates, ibuprofen, mefenamicacid, piroxicam, nabumetone, rofecoxib, celecoxib, etodolac ornimesulide). For the treatment of lupus erythmatosus, for example, thecompounds disclosed herein may also be administered in conjunction withanti-malarial drugs including, for example, hydroxychloroquinone or inconjunction with cytotoxic chemotherapies including, for example,azathioprine and cyclophosphamide.

In some embodiments, the other therapeutic agent is an antibiotic (e.g.,vancomycin, penicillin, amoxicillin, ampicillin, cefotaxime,ceftriaxone, cefixime, rifampinmetronidazole, doxycycline orstreptomycin). In another embodiment, the other therapeutic agent is aPDE4 inhibitor (e.g., roflumilast or rolipram). In another embodiment,the other therapeutic agent is an antihistamine (e.g., cyclizine,hydroxyzine, promethazine or diphenhydramine). In another embodiment,the other therapeutic agent is an anti-malarial (e.g., artemisinin,artemether, artsunate, chloroquine phosphate, mefloquine hydrochloride,doxycycline hyclate, proguanil hydrochloride, atovaquone orhalofantrine).

Another type of therapeutic agent useful in the combination treatment ofthe disclosure is an antibody such as a humanized monoclonal antibody.Non-limiting examples include, the anti-CD99 antibody. See, for example,U.S. Pat. No. 7,223,395; White et al., Annu. Rev. Med., 52:125 (2001).Rituximab (Rituxan®; Genentech, South San Francisco, Calif.) is anothertherapeutic agent that is useful in a conjugate of the disclosure fortreating rheumatoid arthritis. Another therapeutic agent useful in thedisclosure also can be cytotoxic agents, which, as used herein, is anymolecule that directly or indirectly promotes cell death. Specificanticancer agents include Flavopiridol, Adriamycin (doxorubicin), VP16(Etoposide), Taxol (paclitaxel), cisplatin and the like.

In cases where compounds are sufficiently basic or acidic to form stablenontoxic acid or base salts, administration of the compounds as saltsmay be appropriate. Examples of pharmaceutically acceptable salts areorganic acid addition salts formed with acids which form a physiologicalacceptable anion, for example, tosylate, methanesulfonate, acetate,citrate, malonate, tartarate, succinate, benzoate, ascorbate,a-ketoglutarate, and a.-glycerophosphate. Suitable inorganic salts mayalso be formed, including hydrochloride, sulfate, nitrate, bicarbonate,and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

The compounds useful in practicing the disclosure can be formulated aspharmaceutical compositions and administered to a mammalian host, suchas a human patient in a variety of forms adapted to the chosen route ofadministration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

The compounds may be systemically administered, e.g., orally, incombination with a pharmaceutically acceptable vehicle such as an inertdiluent or an assimilable edible carrier. The route of administration isoral or intravenous. Other routes of administration include, forexample, parental, intramuscular, topical and subcutaneous. Thecompounds may be enclosed in hard or soft shell gelatin capsules, may becompressed into tablets, or may be incorporated directly with the foodof the patient's diet. For oral therapeutic administration, the activecompound may be combined with one or more excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 0.1% of active compound. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of a given unit dosage form. The amount of active compound insuch therapeutically useful compositions is such that an effectivedosage level will be obtained.

Just as in case of the compounds of the general structure A, thecompounds of the general structure B may be administered in a variety ofways. For example, the tablets, troches, pills, capsules, and the likemay also contain the following: binders such as gum tragacanth, acacia,corn starch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, fructose, lactose or aspartame or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring may beadded. When the unit dosage form is a capsule, it may contain, inaddition to materials of the herein type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may beas coatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Any material used in preparing any unit dosageform should be pharmaceutically acceptable and substantially non-toxicin the amounts employed. In addition, the active compound may beincorporated into sustained-release preparations and devices.

The compounds may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powders bythe active ingredient which are adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions,optionally encapsulated in liposomes. In all cases, the ultimate dosageform should be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium including, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be advisable to include isotonicagents, for example, sugars, buffers or sodium chloride. Prolongedabsorption of the injectable compositions can be brought about by theuse in the compositions of agents delaying absorption, for example,aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated herein, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the methods of preparation are vacuumdrying and the freeze drying techniques, which yield a powder of theactive ingredient plus any additional desired ingredient in thepreviously sterile-filtered solutions.

For topical administration, the compounds may be applied in pure form,i.e., when they are liquids. However, it will generally be desirable toadminister them to the skin as compositions or formulations, incombination with a dermatologically acceptable carrier, which may be asolid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the compounds of structures A or B to the skin are known in theart; for example, see U.S. Pat. Nos. 4,608,392, 4,992,478, 4,559,157,and 4,820,508.

Useful dosages of the compounds can be determined by comparing their invitro activity, and in vivo activity in animal models. Methods for theextrapolation of effective dosages in mice, and other animals, to humansare known to the art; for example, see U.S. Pat. No. 4,938,949.

Generally, the concentration of the compounds of the general structure Bin a liquid composition, such as a lotion, may be between about 0.1 andabout 25.0 mass %, such as between about 0.5 about 10.0 mass %. Theconcentration in a semi-solid or solid composition such as a gel or apowder may be between about 0.1 and about 5.0 mass %, such as betweenabout 0.5 and 2.5 mass %.

The amount of the compound, or an active salt or derivative thereof,required for use in treatment will vary with the particular saltselected but also with the route of administration, the nature of thecondition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician. In general, however, a suitable dose may be in the range ofbetween about 0.2 and about 100.0 μmol/kg per day. In one embodiment,the dose can be, e.g., between about 0.2 to about 1.0 μmol/kg per day.In some embodiments, a suitable does may be in the rage of between about0.5 and about 100 mg/kg, e.g., between about 10 and about 75 mg/kg ofbody weight per day, such as between about 3 and about 50 mg perkilogram body weight of the recipient per day, for example, in the rangeof between about 6 and about 90 mg/kg/day, such as in the range ofbetween about 15 and about 60 mg/kg/day.

Pharmaceutical compositions suitable for use in the methods disclosedherein include compositions where the active ingredients are containedin an amount effective to achieve its intended purpose. Morespecifically, a therapeutically effective amount means an amount ofcompound effective to prevent, alleviate or ameliorate symptoms ofdisease or prolong the survival of the subject being treated.Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

The exact formulation, route of administration and dosage for thepharmaceutical compositions disclosed herein can be chosen by theindividual physician in view of the patient's condition. Typically, thedose range of the composition administered to the patient can be betweenabout 0.5 and about 1000 mg/kg of the patient's body weight, or betweenabout 1 and about 500 mg/kg, or between about 10 and about 500 mg/kg, orbetween about 50 and about 100 mg/kg of the patient's body weight. Thedosage may be a single one or a series of two or more given in thecourse of one or more days, as is needed by the patient. Where no humandosage is established, a suitable human dosage can be inferred from ED₅₀or ID₅₀ values, or other appropriate values derived from in vitro or invivo studies, as qualified by toxicity studies and efficacy studies inanimals.

Although the exact dosage will be determined on a drug-by-drug basis, inmost cases, some generalizations regarding the dosage can be made. Thedaily dosage regimen for an adult human patient may be, for example, anoral dose of between about 0.1 mg and about 500 mg of each ingredient,such as between about 1 mg and about 250 mg, e.g. between about 5 andabout 200 mg or an intravenous, subcutaneous, or intramuscular dose ofeach ingredient between about 0.01 mg and about 100 mg, such as betweenabout 0.1 mg and about 60 mg, e.g. between about 1 and about 40 mg ofeach ingredient of the pharmaceutical compositions disclosed herein or apharmaceutically acceptable salt thereof calculated as the free base,the composition being administered 1 to 4 times per day. Alternativelythe compositions disclosed herein may be administered by continuousintravenous infusion, at a dose of each ingredient up to 400 mg per day.Thus, the total daily dosage by oral administration of each ingredientwill typically be in the range between about 1 and about 2000 mg and thetotal daily dosage by parenteral administration will typically be in therange between about 0.1 and about 400 mg. In some embodiments, thecompounds will be administered for a period of continuous therapy, forexample for a week or more, or for months or years.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety, which are sufficient to maintain themodulating effects, or minimal effective concentration (MEC). The MECwill vary for each compound but can be estimated from in vitro data.Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. However, HPLC assays orbioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compositionsshould be administered using a regimen, which maintains plasma levelsabove the MEC for 10-90% of the time, such as between 30-90%, e.g.,between 50-90%. In cases of local administration or selective uptake,the effective local concentration of the drug may not be related toplasma concentration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

In various embodiments, the compositions may, if desired, be ed in apack or dispenser device, which may contain one or more unit dosageforms containing the active ingredient. The pack may for examplecomprise metal or plastic foil, such as a blister pack. The pack ordispenser device may be accompanied by instructions for administration.The pack or dispenser may also be accompanied with a notice associatedwith the container in form prescribed by a governmental agencyregulating the manufacture, use, or sale of pharmaceuticals, whichnotice is reflective of approval by the agency of the form of the drugfor human or veterinary administration. Such notice, for example, may bethe labeling approved by the U.S. Food and Drug Administration forprescription drugs, or the approved product insert. Compositionsincluding a compound disclosed herein formulated in a compatiblepharmaceutical carrier may also be prepared, placed in an appropriatecontainer, and labeled for treatment of an indicated condition.

In various embodiments, compounds of the disclosure can be labeled usingmethods known in the art. One detectable group is a fluorescent group.Fluorescent groups typically produce a high signal to noise ratio,thereby providing increased resolution and sensitivity in a detectionprocedure. For example, the fluorescent group absorbs light with awavelength above about 300 nm, such as above about 350 nm, e.g., aboveabout 400 nm. The wavelength of the light emitted by the fluorescentgroup is above about 310 nm, such as above about 360 nm, e.g., aboveabout 410 nm.

The fluorescent detectable moiety can be selected from a variety ofstructural classes, including the following non-limiting examples: 1-and 2-amino-naphthalene, p,p′ diaminostilbenes, pyrenes, quaternaryphenanthridine salts, 9-aminoacridines, p,p′-diaminobenzophenone imines,anthracenes, oxacarbocyanine, marocyanine, 3-aminoequilenin, perylene,bisbenzoxazole, bis-p-oxazolyl benzene, 1,2-benzophenazin, retinol,bis-3-aminopridinium salts, hellebrigenin, tetracycline, sterophenol,benzimidazolyl phenylamine, 2-oxo-3-chromen, indole, xanthen,7-hydroxycoumarin, phenoxazine, salicylate, strophanthidin, porphyrins,triarylmethanes, flavin, xanthene dyes (e.g., fluorescein and rhodaminedyes); cyanine dyes; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dyes andfluorescent proteins (e.g., green fluorescent protein,phycobiliprotein).

In various embodiments, the compounds can be labeled, where the labelinggroup spontaneously emits a signal, or generates a signal upon theintroduction of a suitable stimulus. Labels, include atoms such as, forexample, ¹³C, ¹⁵N, ¹⁹F, ¹H and the like. In various embodiments, thecompound can be conveniently administered in unit dosage form; forexample, containing between about 5 and about 1,000 mg, such as betweenabout 10 and about 750 mg, e.g., between about 50 and about 500 mg ofactive ingredient per unit dosage form.

In some embodiments, the active ingredient can be administered toachieve peak plasma concentrations of the active compound of betweenabout 0.5 and about 75 μM, such as between about 1 and about 50 μM,e.g., between about 2 and about 30 μM. This may be achieved, forexample, by the intravenous injection of a 0.05 to 5% solution of theactive ingredient, optionally in saline, or orally administered as abolus containing about 1-100 mg of the active ingredient. Desirableblood levels can be maintained by, for example, continuous infusion toprovide about 0.01-5.0 mg/kg/hr or by intermittent infusions containingabout 0.4-15 mg/kg of the active ingredient(s).

The desired dose may conveniently be ed in a single dose or as divideddoses administered at appropriate intervals, for example, as two, three,four or more sub-doses per day. The sub-dose itself may be furtherdivided, e.g., into a number of discrete loosely spaced administrations;such as multiple inhalations from an insufflator.

EXAMPLES

Some aspects of the disclosure can be further illustrated by thefollowing non-limiting examples.

Example 1 Molecular Modeling

Molecular modeling studies were conducted on a Linux workstation and a64 3.2-GHz CPUs Linux cluster. Docking studies were performed using thecrystal structure of BCL-X_(L) in complex with Bak-derived peptide(Protein Data Bank code 1BXL). The docked structures of 5, 5′substituted Apogossypol derivatives in the peptide-binding pocket wereobtained by ChemScore as the scoring function in the GOLD dockingprogram. The protein surface was prepared with the program MOLCAD asimplemented in Sybyl (Tripos, St. Louis). Docking studies were alsoperformed using the crystal structure of Bcl-2 in complex with abenzothiazole BH3 mimetic ligand (Protein Data Bank code 1YSW). Theligand was extracted from the protein structure and was used to definethe binding site for small molecules. Apogossypol and its derivativeswere docked into the Bcl-2 protein by the GOLD docking program usingChemScore as the scoring function. The active site radius was set at 10Å and 10 GA solutions were generated for each molecule. The GA dockingprocedure in GOLD allowed the small molecules to flexibly explore thebest binding conformations whereas the protein structure was static. Theprotein surface was prepared with the program MOLCAD⁵ as implemented inSybyl (Tripos, St. Louis) and was used to analyze the binding poses forstudied small molecules.

Example 2 General Chemical Procedures

Unless otherwise noted, all reagents and anhydrous solvents (CH₂Cl₂,THF, diethyl ether, etc) were obtained from commercial sources and usedwithout purification. All reactions were performed in oven-driedglassware. All reactions involving air or moisture sensitive reagentswere performed under a nitrogen atmosphere. Silica gel or reverse phasechromatography was performed using prepacked silica gel or C-18cartridges (RediSep), respectively. All final compounds were purifiedto >95% purity, as determined by a HPLC Breeze from Waters Co. using anAtlantis T3 3 μM 4.6 mm×150 mm reverse phase column. Compounds for invivo studies were purified again using preparative HPLC again to >99%purity. The eluant was a linear gradient with a flow rate of 1 ml/minfrom 50% A and 50% B to 5% A and 95% B in 15 min followed by 5 min at100% B (Solvent A: H₂O with 0.1% TFA; Solvent B: ACN with 0.1% TFA).Compounds were detected at λ=254 nm. NMR spectra were recorded on Varian300 or Bruker 600 MHz instruments. Chemical shifts are reported in ppm(δ) relative to ¹H (Me₄Si at 0.00 ppm). Coupling constant (J) arereported in Hz throughout. Mass spectral data were acquired on ShimadzuLCMS-2010EV for low resolution, and on an Agilent ESI-TOF for eitherhigh or low resolution.

Example 3 Synthesis of Compounds of the Disclosure

The synthesis for 5,5′ substituted apogossypol derivatives is outlinedbelow:

Briefly and generally, gossypol 1 was treated with NaOH solutionfollowed by dimethyl sulfate to afford methyl apogossypol. Reaction ofmethyl apogossypol with TiCl₄ and dichloromethyl methyl ether resultedin loss of isopropyl groups and simultaneous bisformylation to givealdehyde 2. The compound 2 was treated with different Grignard orlithium reagents to afford a secondary alcohol, which was oxidized tothe phenone using pyridinium chlorochromate. Subsequent demethylation ofthe phenone afforded compound 3.

More specifically, the gossypol acetic acid 1 (5 g, 8.65 mmol) in 50 mlof 40% NaOH was heated under nitrogen at 90° C. for 3.5 hours in thedark. The reaction mixture was cooled and poured slowly onto ice (300ml) and concentrated H₂SO₄ (35 ml) mixture to form white precipitation.The precipitation was filtered, washed with water and dried to affordapogossypol (3.8 g, 95%) as a white solid. ¹H NMR (CDCl₃ δ 7.61 (s, 2H),7.50 (s, 2H), 5.93 (s, 2H), 5.27 (s, 2H), 5.13 (s, 2H), 3.88 (m, 2H),2.12 (s, 6H), 1.55 (d, J=5.5 Hz, 12H).

Apogossypol (3.8 g, 8.21 mmol) was then dissolved into 200 ml acetone.K₂CO₃ (23.9 g, 206.7 mmol) and dimethyl sulfate (16.3 ml, 206.7 mmol)were added and the reaction mixture was refluxed under nitrogen for 24hours. The solid that separated from the solution was collected byfiltration. It was washed (acetone and water) and dried to yield 4.2 gof methylated apogossypol (93%). To a solution of methylated apogossypol(1.6 g, 2.93 mmol) in dry methyl chloride (40 ml) at 0° C. was addedtitanium tetrachloride (14.3 g, 75.5 mmol). After addition wascompleted, the dark red solution was stirred an additional 15 min at 0°C. Dichloromethyl methyl ether (2.93 g, 25.5 mmol) was added dropwiseover 15 min, and the reaction mixture was stirred at ambient temperatureunder nitrogen for 14 hr.

The reaction mixture was poured onto ice and the resulting aqueous layerwas extracted twice with methyl chloride. The combined organic fractionswere washed with water and brine, dried over MgSO₄, and concentrated togive dark red oil. The oil was chromatographed (acetonitrile/methylchloride) followed by trituration of crude product with diethyl ether toafford intermediate 2 (0.60 g, 40%) as a yellow solid.

For intermediate 2: ¹H NMR: 8.47 (s, 1H), 7.29 (s, 1H), 7.05 (br s, 1H),2.79 (t, J=7.35 Hz, 2H), 2.47 (s, 3H), 2.44 (s, 3H), 1.70 (m, 2H), 1.03(t, J=7.35 Hz, 3H).

Example 4 Synthesis of Compound I of the Disclosure

Compound I of the disclosure having the formula shown herein, also knownas1,1′-(1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-diyl)bis(2-phenylethanone),was synthesized as follows. To a freshly benzylmagnesium chloride (5.4mmol) solution at room temperature was added a solution of aldehyde 2(1.0 g, 1.93 mmol) in anhydrous tetrahydrofuran (15 ml) and the reactionmixture was stirred at this temperature for 12 hr. The reaction mixturewas poured onto saturated ammonium chloride solution and the aqueouslayer was extracted twice with diethyl ether, washed with brine anddried over MgSO₄. Filtration followed by evaporation of the ether gaveyellow oil. The solution of yellow oil in dry methyl chloride (10 ml)was added into pyridinium chlorochromate (2.6 g, 12.1 mmol) in drymethyl chloride (12 ml). The reaction mixture was stirred at ambienttemperature for 4 hr and was filtrated through celite

The filtrate was chromatographed to afford 0.3 g of methylated compoundI (22%). 0.27 mL of BBr₃ solution (0.72 g, 2.88 mmol) was added dropwiseinto a solution of methylated compound I (120 mg, 0.17 mmol) in 8 mL ofanhydrous CH₂Cl₂ at −78° C. Stirring was continued at −78° C. for 1 hr,0° C. for 1 hr, and ambient temperature for 1 hr. 50 grams of icecontaining 10 mL of 6M hydrochloric acid was added to the mixture andstirred for one hour at room temperature. The aqueous layer wasextracted with dichloromethane (3×30 mL). The combined organic layer waswashed with water, brine and dried over MgSO₄. The solvent wasconcentrated in vacuo and the residue was purified by C-18 columnchromatography (H₂O/Acetonitrile) to give 80 mg of compound c I (77%) asorange solid.

¹H NMR (CD₃OD) δ 7.61 (s, 2H), 7.30 (m, 8H), 7.22 (m, 2H), 6.97 (s, 2H),4.40 (dd, J₁=15.6 Hz, J₂=22.8 Hz, 4H), 1.87 (s, 6H); ¹³C NMR (CD₃)₂SO) δ204.6, 149.4, 144.8, 144.5, 135.4, 134.2, 130.5, 128.6, 126.9, 126.3,122.6, 119.4, 116.8, 115.0, 107.1, 51.0, 21.1; HRMS calcd for[C₃₈H₃₀O₈+H] 615.2019; found 615.2013. HPLC is 99% pure.

Other derivatives encompassed by general structure A were synthesizedand characterized. The synthesis followed the pattern described inExamples 3 and 4, with necessary adjustments, such as using differentGrignard or lithium reagents when treating aldehyde intermediatecompound 2. The spectral characteristics of the compounds were asfollows (Roman numerals correspond to the herein described compounds ofthe disclosure).

Compound III.1,1′-(1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-diyl)bis(2-methylpropan-1-one).¹H NMR (CDCl₃) δ 12.38 (s, 2H), 7.99 (s, 2H), 7.82 (s, 2H), 7.44 (s,2H), 6.18 (s, 2H), 5.41 (s, 2H), 3.86 (m, 2H), 2.13 (s, 6H), 1.33 (d,J=9 Hz, 12H).

Compound XVIII.1,1′-(1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-diyl)bis(2,2-dimethylpropan-1-one).¹H NMR (CD₃OD) δ 7.56 (s, 2H), 6.78 (s, 2H), 1.95 (s, 6H), 1.34 (m,18H).

Compound IV.1,1′-(1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-diyl)bis(3-methylbutan-1-one).¹H NMR (CD₃OD) δ 7.62 (s, 2H), 7.12 (s, 2H), 2.97 (d, J=6.6 Hz, 4H),2.32 (m, 2H), 1.96 (s, 6H), 1.03 (d, J=3.6 Hz, 12H).

Compound XX.1,1′-(1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-diyl)dipentan-1-one.¹H NMR (CD₃OD) δ 7.62 (s, 2H), 7.07 (s, 2H), 3.07 (t, J₁=J₂=6.6 Hz, 4H),1.97 (s, 6H), 1.76 (m, 4H), 1.45 (m, 4H), 0.97 (t, J₁=J₂=6.6 Hz, 6H).

Compound XIX.1,1′-(1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-diyl)bis(2-methylbutan-1-one).¹H NMR (CD₃OD) δ 7.62 (s, 2H), 7.05 (s, 2H), 3.43 (m, 2H), 1.96 (s, 6H),1.50 (m, 4H), 1.21 (d, J=6.6 Hz, 6H), 0.99 (d, J=7.2 Hz, 6H).

Compound VII.(1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-diyl)bis(phenylmethanone).¹H NMR (CD₃OD) δ 7.89 (d, J=6.6 Hz, 4H), 7.67 (s, 2H), 7.62 (s, 2H),7.49 (s, 4H), 6.82 (s, 2H), 1.93 (s, 6H).

Compound XVII.(1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-diyl)bis(benzo[d]thiazol-2-ylmethanone).¹H NMR (CD₃OD) δ 8.14 (d, J=4.8 Hz, 2H), 8.07 (s, 2H), 7.75 (s, 2H),7.59 (t, J₁=J₂=2.4 Hz, 4H), 7.03 (s, 2H), 1.93 (s, 6H).

Compound VI.(1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-diyl)bis(cyclopentylmethanone).¹H NMR (CD₃OD) δ 7.62 (s, 2H), 7.05 (s, 2H), 3.84 (m, J₁=J₂=7.2 Hz, 2H),2.03 (m, 4H), 1.99 (s, 6H), 1.93 (m, 4H), 1.77 (m, 4H), 1.67 (m, 4H).

Compound VIII.(1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-diyl)bis(naphthalen-1-ylmethanone).¹H NMR (CD₃OD) δ 8.97 (d, J=7.8 Hz, 2H), 8.07 (m, 2H), 7.98 (d, J=7.8Hz, 2H), 7.68 (m, 8H), 7.43 (m, 2H), 6.95 (s, 2H), 1.79 (s, 6H).

Compound V.1,1′-(1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-diyl)bis(3-ethylheptan-1-one).¹H NMR ((CD₃)₂SO) δ 10.08 (s, 2H), 9.26 (s, 2H), 8.08 (s, 2H), 7.53 (s,2H), 6.91 (s, 2H), 2.87 (d, J=5.7 Hz, 4H), 1.98 (m, 2H), 1.85 (s, 6H),1.30 (m, 16H), 0.87 (t, J₁=J₂=7.5 Hz, 12H).

Compound IX.(1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-diyl)bis(biphenyl-4-ylmethanone).¹H NMR (CD₃OD) δ 7.97 (d, J=8.1 Hz, 4H), 7.70 (m, 10 H), 7.46 (m, 6H),6.86 (s, 2H), 1.88 (s, 6H).

Compound X.(1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-diyl)bis((4-tert-butylphenyl)methanone).¹H NMR (CD₃OD) δ 7.82 (d, J=8.4 Hz, 4H), 7.65 (s, 2H), 7.51 (d, J=8.4Hz, 4H), 6.80 (s, 2H), 1.86 (s, 6H), 1.34 (s, 18H).

Compound XI.(1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-diyl)bis((4-(trifluoromethyl)phenyl)methanone).¹H NMR (CD₃OD) δ 8.04 (d, J=7.8 Hz, 4H), 7.78 (d, J=7.8 Hz, 4H), 7.69(s, 2H), 6.87 (s, 2H), 1.88 (s, 6H).

Compound II. (3,3′-dimethyl-2,2′-binaphthyl-1,1′,6,6′ 7,7′-hexaol). ¹HNMR (CD₃OD) δ 7.46 (s, 2H), 7.11 (s, 2H), 7.03 (s, 2H), 1.97 (s, 6H).

Compound XVI.1,1′-(1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-diyl)bis(3-(4-fluorophenyl)propan-1-one).¹H NMR (CD₃OD) δ 7.62 (s, 2H), 7.27 (d, J=5.4 Hz, 4H), 6.97 (m, 4H),6.88 (s, 2H), 3.40 (t, J₁=J₂=6.6 Hz, 4H), 3.10 (t, J₁=J₂=6.6 Hz, 4H),1.90 (s, 6H).

Compound XII.1,1′-(1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-diyl)bis(2-p-tolylethanone).¹H NMR (CD₃OD) δ 7.59 (s, 2H), 7.15 (d, J=8.1 Hz, 4H), 7.05 (d, J=8.1Hz, 4H), 6.93 (s, 2H), 4.30 (dd, J₁=15.6 Hz, J₂=9.9 Hz, 4H), 2.27 (s,6H), 1.85 (s, 6H).

Compound XV.1,1′-(1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-diyl)bis(2-cyclohexylethanone).¹H NMR (CD₃OD) δ 7.61 (s, 2H), 7.10 (s, 2H), 2.95 (dd, J₁=3.3 Hz, J₂=3.0Hz, 4H), 2.02 (m, 2H), 1.95 (s, 6H), 1.76 (m, 10H), 1.11 (m, 10H).

Compound XIII.1,1′-(1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-diyl)bis(2-(3-bromophenyl)ethanone).¹H NMR (CD₃OD) δ 7.63 (s, 2H), 7.51 (s, 2H), 7.29 (m, 6H), 7.00 (s, 2H),4.36 (dd, J₁=8.1 Hz, J₂=9.0 Hz, 4H), 1.91 (s, 6H).

Compound XIV.1,1′-(1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-diyl)bis(2-(4-(trifluoromethoxy)phenyl)ethanone).¹H NMR (CD₃OD) δ 7.63 (s, 2H), 7.41 (d, J=4.2 Hz, 4H), 7.20 (d, J=4.2Hz, 4H), 6.99 (s, 2H), 4.40 (dd, J₁=8.1 Hz, J₂=7.2 Hz, 4H), 1.88 (s,6H).

Some compounds of the disclosure may be synthesized as shown on FIG. 12(where R is CONX or CONR₁X, where R or R₁ is an alkyl, aromatic, orheterocyclic group, and X is an alkyl, a substituted alkyl, an aryl, asubstituted aryl, an alkylaryl, and a heterocycle).

Further spectral data and the data on purity with respect to compoundsof the disclosure are summarized in Table 7.

TABLE 7 HIGH RESOLUTION MASS (HRMS) AND HPLC PURITY OF 5,5′ SUBSTITUTEDAPOGOSSYPOL DERIVATIVES Chemical Formula HRMS HPLC Compound [M + H]⁺Calculated Found Purity (%) Gossypol C₃₀H₃₁O₈ NR NR 99.6 ApogossypolC₂₈H₃₁O₆ 463.2115 463.2108 99.5 III C₃₀H₃₁O₈ 519.2013 519.2013 99.5XVIII C₃₂H₃₅O₈ 547.2326 547.2327 99.3 IV C₃₂H₃₅O₈ 547.2326 547.2326 99.3XX C₃₂H₃₅O₈ 547.2326 547.2324 97.1 VII C₃₆H₂₇O₈ 587.1700 587.1702 99.4XVII C₃₈H₂₅N₂O₈S₂ 701.1047 701.1042 97.8 VI C₃₄H₃₅O₈ 571.2326 571.232598.8 VIII C₄₄H₃₁O₈ 687.2013 687.2027 97.2 I C₃₈H₃ ₁O₈ 615.2013 615.201499.0 V C₄₀H₅₁O₈ 659.3578 659.3583 98.8 IX C₄₈H₃₅O₈ 739.2326 739.232399.5 X C₄₄H₄₃O₈ 699.2952 699.2946 99.6 XI C₃₈H₂₅F₆O₈ 723.1448 723.144799.6 II C₂₂H₁₉O₆ 379.1176 379.1168 98.5 XVI C₄₀H₃₃F₂O₈ 679.2138 679.213996.8 XVII C₄₀H₃₅O₈ 643.2326 643.2328 98.6 XV C₃₈H₄₃O₈ 627.2952 627.294998.6 XIII C₃₈H₂₉Br₂O₈ 771.0224 771.0225 98.1 XII C₄₀H₂₉F₆O₁₀ 783.1659783.1651 95.6

Example 5 Synthesis of5,5′-diisopropyl-3,3′-dimethyl-2,2′-binaphthyl-1,1′,6,6′,7,7′-hexanol(2)

The Gossypol acetic acid (1) (5 g, 8.65 mmol) in 50 ml of 40% NaOH washeated under nitrogen at 90° C. for 3.5 hours in the dark. The reactionmixture was cooled and poured slowly onto ice (300 ml) and concentratedH₂SO₄ (35 ml) mixture to form white precipitation. The precipitation wasfiltered, washed with water and dried to afford compound 2 (Apogossypol)(3.8 g, 95%) as a white solid. ¹H NMR (CDCl₃) δ 7.61 (s, 2H), 7.50 (s,2H), 5.93 (s, 2H), 5.27 (s, 2H), 5.13 (s, 2H), 3.88 (m, 2H), 2.12 (s,6H), 1.55 (d, J=5.5 Hz, 12H).

Example 6 Synthesis of5,5′-diisopropyl-1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-2,2′-binaphthyl(4)

The compound 2 (Apogossypol) (3.8 g, 8.21 mmol) was dissolved into 200ml acetone. K₂CO₃ (23.9 g, 206.7 mmol) and dimethyl sulfate (16.3 ml,206.7 mmol) were added and the reaction mixture was refluxed undernitrogen for 24 hours. The solid was collected by filtration and washedusing acetone and water and dried to yield 4.2 g of compound 4 as whitesolid (93%). ¹H NMR (CDCl₃) 7.83 (s, 2H), 7.43 (s, 2H), 3.98 (m, 8H),3.94 (s, 6H), 3.57 (s, 6H), 2.20 (s, 6H), 1.56 (s, 12H).

Example 7 Synthesis of1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarbaldehyde(5)

To a solution of compound 4 (1.6 g, 2.93 mmol) in dry methylene chloride(40 ml) at 0° C. was added titanium tetrachloride (14.3 g, 75.5 mmol).After addition was completed, the dark red solution was stirred anadditional 15 min at 0° C. Dichloromethyl methyl ether (2.93 g, 25.5mmol) was added dropwise over 15 min, and the reaction mixture wasstirred at ambient temperature under nitrogen for 12 hours. The reactionmixture was poured onto ice and the resulting aqueous layer wasextracted twice with methylene chloride. The combined organic fractionswere washed with water and brine, dried over MgSO₄, and concentrated togive dark red oil. The oil was chromatographed (acetonitrile/methylenechloride) followed by trituration of crude product with diethyl ether toafford compound 5 (0.60 g, 40%) as yellow solid. ¹H NMR (CDCl₃): 10.84(s, 2H), 8.93 (s, 2H), 7.82 (s, 2H), 4.10 (s, 6H), 4.03 (s, 6H), 3.48(s, 6H), 2.22 (s, 6H).

Example 8 Synthesis of1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarboxylicacid (6)

Compound 5 (6.6 g, 12.7 mmol) was dissolved in 40 ml of acetonitrile and40 ml of THF in an ice bath. Sodium dihydrogen phosphate (876 mg, 6.35mmol), 30% hydrogen peroxide (2.6 mL, 25.4 mmol) were added. Sodiumchlorite (4.14 g, 45.8 mmol) dissolved in 20 ml of water was added. Thereaction mixture was stirred overnight at room temperature and thenpoured onto 100 g of ice with 30 ml of 6M HCl. The solution wasextracted with ether (3×100 mL). The ether extracts were washed withbrine, dried over magnesium sulfate and filtered. Evaporation of thesolvent in vacuo and the residue was purified by C-18 columnchromatography (H₂O/Acetonitrile) to give 5.9 g (85%) of compound 6 as ared solid. ¹H NMR (CD₃₀D) δ 8.0 (s, 2H), 7.68 (s, 2H), 4.1 (s, 6H), 4.06(s, 6H), 3.54 (s, 6H), 2.21 (s, 6H).

Example 9 Synthesis of1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-N⁵,N^(5′)-bis(2-phenylpropyl)-2,2′-binaphthyl-5,5′-dicarboxamide(7r)

Compound 6 (500 mg, 0.907 mmol), EDCI (522 mg, 2.72 mmol) and HOBT (244mg, 1.81 mmol) were dissolved in 15 ml of dry CH₂Cl₂ and stirred at roomtemperature for 10 minutes under nitrogen atmosphere.2-phenyl-1-propanamine (0.30 ml, 2.09 mmol) and Et₃N (0.51 ml, 3.7 mmol)were added and the reaction mixture was stirred at room temperature for24 hours. The mixture was then poured onto 50 ml of water and thesolution was extracted with CH₂Cl₂ (3×100 mL). The ether extracts werewashed with water and brine, dried over magnesium sulfate and filtered.Evaporation of the solvent in vacuo and the residue was purified bysilica chromatography to give 320 mg (45%) of compound 7r as a yellowsolid. ¹H NMR (CD₃OD) δ 7.56 (s, 2H), 7.37 (m, 8H), 7.22 (m, 4H), 3.98(s, 6H), 3.85 (s, 6H), 3.77 (m, 2H), 3.62 (m, 2H), 3.55 (s, 3H), 3.53(s, 3H), 3.20 (m, 2H), 2.01 (s, 3H), 2.00 (s, 3H), 1.38 (s, 3H), 1.39(s, 3H).

Example 10 Synthesis of1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-N⁵,N^(5′)-bis(2-phenylpropyl)-2,2′-binaphthyl-5,5′-dicarboxamide(8r)

0.45 ml of BBr₃ solution (1.18 g, 4.73 mmol) was added dropwise into asolution of compound 7 (310 mg, 0.40 mmol) in 20 ml of anhydrous CH₂Cl₂at −78° C. Stirring was continued at −78° C. for 1 hour, 0° C. for 2hours, and ambient temperature for 10 minutes. 50 grams of icecontaining 10 ml of 6M HCl was added to the mixture and stirred for onehour at room temperature. The aqueous layer was extracted withdichloromethane (3×50 ml). The combined organic layer was washed withwater, brine and dried over MgSO₄, The solvent was concentrated in vacuoand the residue was purified using C-18 column chromatography(H₂O/Acetonitrile) to give 200 mg of compound 8r (72%) as white-yellowsolid. ¹H NMR (CD₃OD) δ 7.56 (s, 2H), 7.37 (d, J=6.0 Hz, 4H), 7.32 (t,J₁=J₂=7.2 Hz, 4H), 7.20 (t, J₁=J₂=7.2 Hz, 2H), 7.06 (s, 1H), 6.98 (s,1H), 3.75-3.59 (m, 4H), 3.19 (m, 2H), 1.88 (d, J=3.0 Hz, 3H), 1.88 (d,J=3.0 Hz, 3H), 1.41 (m, 6H).

Following the herein mentioned procedure and the appropriate startingmaterials and reagents used; compounds (7a-7t, 8a-8t and 14) weresynthesized.

Example 11 Synthesis of1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-5,5′-diphenethyl-2,2′-binaphthyl(11e)

To a freshly benzylmagnesium chloride (5.4 mmol) solution at roomtemperature was added a solution of 5 (1.0 g, 1.93 mmol) in anhydroustetrahydrofuran (15 ml) and the reaction mixture was stirred at thistemperature for 12 hours. The reaction mixture was poured onto saturatedammonium chloride solution and the aqueous layer was extracted twicewith diethyl ether, washed with brine and dried over MgSO₄. Filtrationfollowed by evaporation of the ether gave yellow oil. The solution ofyellow oil in dry methylene chloride (10 ml) was added into pyridiniumchlorochromate (2.6 g, 12.1 mmol) in dry methylene chloride (12 ml). Thereaction mixture was stirred at ambient temperature for 4 hours and wasfiltrated through celite. The filtrate was chromatographed to afford 0.3g of 10e (22%). ¹H NMR (CDCl₃) δ 7.54 (s, 2H), 7.32 (m, 10H), 7.14 (s,2H), 4.29 (s, 4H), 4.02 (s, 6H), 3.96 (s, 6H), 3.49 (s, 6H), 2.02 (s,6H). To a solution of compound 10e (170 mg, 0.29 mmol) in 10 ml TFA wasadded 0.6 ml of triethylsilane dropwise. The solution was stirredovernight at room temperature and concentrated in vacuo followed bysilica gel column chromatography to give compound 11e as colorless oil(140 mg, 90%). ¹H NMR (CDCl₃) δ 7.67 (s, 2H), 7.44 (s, 2H), 7.35 (s,8H), 7.25 (s, 2H), 4.04 (s, 6H), 3.95 (s, 6H), 3.60 (s, 6H), 3.41 (m,4H), 3.02 (m, 4H), 2.18 (s, 6H).

Example 12 Synthesis of3,3′-dimethyl-5,5′-diphenethyl-2,2′-bipnaphthyl-1,1′,6,6′,7,7′-hexaol(12e)

0.27 ml of BBr₃ solution (0.72 g, 2.88 mmol) was added dropwise into asolution of 11e (200 mg, 0.30 mmol) in 8 ml of anhydrous CH₂Cl₂ at −78°C. Stirring was continued at −78° C. for 1 hour, 0° C. for 1 hour, andambient temperature for 1 hour, respectively. 100 grams of icecontaining 10 ml of 6M HCl was added to the mixture and stirred for onehour at room temperature. The aqueous layer was extracted withdichloromethane (3×50 ml). The combined organic layer was washed withwater, brine and dried over MgSO₄. The solvent was concentrated in vacuoand the residue was purified by C-18 column chromatography(H₂O/Acetonitrile) to give 128 mg of compound 12e (75%) as orange solid.¹H NMR (CDCl₃) δ 7.52 (s, 2H), 7.44 (s, 2H), 7.30 (m, 10H), 5.35 (s, OH,4H), 5.17 (s, OH, 2H), 3.37 (t, J₁=J₂=6.6 Hz, 4H), 3.03 (t, J₁=J₂=6.6Hz, 4H), 2.13 (s, 6H).

Example 13 Synthesis of compounds 11a-11e and 12a-12e

Following herein mentioned procedure and the appropriate startingmaterials and reagents used; compounds (11a-11e and 12a-12e) weresynthesized.

1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-N⁵,N⁵′-diphenyl-2,2′-binaphthyl-5,5′-dicarboxamide(7a). Yield, 45%. ¹H NMR (CD₃OD) δ 7.76 (d, J=7.8 Hz, 4H), 7.59 (s, 2H),7.52 (s, 2H), 7.40 (t, J₁=J₂=7.8 Hz, 4H), 7.18 (s, 2H), 4.04 (s, 6H),4.00 (s, 6H), 3.63 (s, 6H), 2.11 (s, 6H).

N⁵,N⁵′-dicyclopentyl-1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarboxamide(7b). Yield, 40%. ¹H NMR (CD₃OD) δ 7.52 (s, 2H), 7.45 (s, 2H), 4.47 (m,2H), 3.98 (s, 6H), 3.96 (s, 6H), 3.60 (s, 6H), 2.11 (m, 10H), 1.79 (s,4H), 1.68 (s, 8H).

1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-N⁵,N⁵′-bis(4-phenoxyphenyl)-2,2′-binaphthyl-5,5′-dicarboxamide(7c). Yield, 46%. ¹H NMR (CD₃OD) δ 7.76 (m, 6H), 7.59 (m, 2H), 7.53 (m,2H), 7.35 (m, 2H), 7.11 (m, 2H), 7.03 (m, 8H), 4.00 (s, 6H), 4.00 (s,6H), 3.63 (s, 6H), 2.12 (s, 6H).

N⁵,N⁵′-bis(3-ethylphenyl)-1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarboxamide(7d). Yield, 47%. ¹H NMR (CD₃OD) δ 7.62 (s, 2H), 7.58 (m, 4H), 7.52 (s,2H), 7.30 (m, 2H), 7.05 (m, 2H), 4.04 (s, 6H), 3.99 (s, 6H), 3.63 (s,6H), 2.54 (q, J₁=J₂=8.4 Hz, 4H), 2.11 (s, 6H), 1.28 (t, J₁=J₂=8.4 Hz,6H).

1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-N⁵,N⁵′-bis(3-(trifluoromethyl)phenyl)-2,2′-binaphthyl-5,5′-dicarboxamide(7e). Yield, 50%. ¹H NMR (CD₃OD) δ 7.88 (s, 2H), 7.77 (d, J=6.6 Hz, 2H),7.60 (m, 4H), 7.54 (s, 2H), 7.36 (s, 2H), 4.77 (s, 4H), 3.99 (s, 6H),3.94 (s, 6H), 3.58 (s, 6H), 2.05 (s, 6H).

1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-N5,N5′-bis(1-phenylpropyl)-2,2′-binaphthyl-5,5′-dicarboxamide(7f). Yield, 46%. ¹H NMR (CD₃OD) δ 7.50 (m, 6H), 7.38 (m, 4H), 7.26 (m,4H), 5.12 (s, 2H), 4.01 (s, 6H), 4.00 (s, 6H), 3.89 (s, 6H), 3.58 (s,3H), 3.55 (s, 3H), 1.95 (m, 10H), 1.10 (s, 6H).

N⁵,N⁵′-dibenzyl-1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarboxamide(7g). Yield, 46%. ¹H NMR (CD₃OD) δ 7.53 (m, 6H), 7.38 (m, 6H), 7.30 (m,2H), 4.68 (s, 4H), 4.00 (s, 6H), 3.91 (s, 6H), 3.57 (s, 6H), 2.02 (s,6H).

1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-N⁵,N⁵′-bis(3-methylbenzyl)-2,2′-binaphthyl-5,5′-dicarboxamide(7h). Yield, 43%. ¹H NMR (CD₃OD) δ 7.52 (s, 2H), 7.36 (d, J=7.8 Hz, 4H),7.29 (d, J=7.8 Hz, 2H), 7.26 (t, J₁=7.8 Hz, J₂=7.2 Hz, 2H), 7.11 (d,J=7.2 Hz, 2H), 4.64 (s, 4H), 4.00 (s, 6H), 3.92 (s, 6H), 3.57 (s, 6H),2.37 (s, 6H), 2.02 (s, 6H).

N⁵,N⁵′-bis(3-chlorobenzyl)-1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarboxamide(7i). Yield, 46%. ¹H NMR (CD₃OD) δ 7.61 (s, 2H), 7.53 (s, 2H), 7.42 (d,J=6.6 Hz, 2H), 7.36 (m, 4H), 7.31 (d, J=7.2 Hz, 2H), 4.68 (s, 4H), 4.00(s, 6H), 3.98 (s, 6H), 3.58 (s, 6H), 2.07 (s, 6H).

1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-N⁵,N⁵′-bis(2,4,6-trimethylbenzyl)-2,2′-binaphthyl-5,5′-dicarboxamide(7j). Yield, 40%. ¹H NMR (CD₃OD) δ 7.48 (s, 2H), 7.41 (s, 2H), 6.96 (s,2H), 6.88 (s, 2H), 3.92 (s, 6H), 3.87 (s, 6H), 3.55 (s, 6H), 3.39 (s,6H), 2.46 (s, 6H), 2.27 (s, 6H), 2.05 (s, 6H).

N⁵,N⁵′-bis(1-(4-chlorophenyl)ethyl)-1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarboxamide(7k). Yield, 49%. ¹H NMR (CD₃OD) δ 7.52 (m, 6H), 7.39 (m, 4H), 7.24 (s,1H), 7.25 (s, 1H), 5.36 (m, 2H), 4.01 (s, 3H), 4.00 (s, 3H), 3.90 (s,3H), 3.89 (s, 3H), 3.57 (s, 3H), 3.56 (s, 3H), 2.01 (s, 3H), 2.00 (s,3H), 1.58 (s, 3H), 1.57 (s, 3H).

N⁵,N⁵′-bis(cyclopropylmethyl)-1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarboxamide(7l). Yield, 46%. ¹H NMR (CD₃OD) δ 7.52 (s, 2H), 7.49 (s, 2H), 4.00 (s,6H), 3.96 (s, 6H), 3.59 (s, 6H), 3.37 (d, J=6.9 Hz, 4H), 2.10 (s, 6H),1.2 (m, 2H), 0.59 (m, 4H), 0.37 (m, 4H).

N⁵,N⁵′-bis(cyclohexylmethyl)-1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarboxamide(7m). Yield, 50%. ¹H NMR (CD₃OD) δ 7.52 (s, 2H), 7.45 (s, 2H), 4.02 (s,6H), 3.94 (s, 6H), 3.59 (s, 6H), 3.33 (d, J=17.4 Hz, 4H), 2.09 (s, 6H),1.94 (d, J=12.0 Hz, 4H), 1.80 (d, J=12.0 Hz, 4H), 1.72 (d, J=10.6 Hz,4H), 1.39-1.07 (m, 10H).

1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-N⁵,N⁵′-diphenethyl-2,2′-binaphthyl-5,5′-dicarboxamide(7n). Yield, 51%. ¹H NMR (CD₃OD) δ 7.51 (s, 2H), 7.36 (d, J=7.2 Hz, 4H),7.30 (m, 6H), 7.22 (t, J₁=J₂=7.2 Hz, 2H), 4.00 (s, 6H), 3.89 (s, 6H),3.78 (t, J₁=7.2 Hz, J₂=6.6 Hz, 4H), 3.57 (s, 6H), 3.02 (t, J₁=6.6 Hz,J₂=7.2 Hz, 4H), 2.04 (s, 6H).

1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-N⁵,N⁵′-bis(3-methylphenethyl)-2,2′-binaphthyl-5,5′-dicarboxamide(7o). Yield, 50%. ¹H NMR (CD₃OD) δ 7.51 (s, 2H), 7.28 (s, 2H), 7.23 (m,4H), 7.12 (m, 4H), 3.97 (s, 6H), 3.89 (s, 6H), 3.75 (s, 6H), 3.58 (m,4H), 2.97 (m, 4H), 2.29 (s, 6H), 2.02 (s, 6H).

N⁵,N⁵′-bis(3-chlorophenethyl)-1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarboxamide(7p). Yield, 45%. ¹H NMR (CD₃OD) δ 7.51 (s, 2H), 7.39 (s, 2H), 7.30 (d,J=4.2 Hz, 4H), 7.25 (m, 4H), 4.03 (s, 6H), 3.95 (s, 6H), 3.78 (m, 4H),3.55 (s, 6H), 3.02 (t, J₁=J₂=6.6 Hz, 4H), 2.04 (s, 6H).

N⁵,N⁵′-bis(4-ethylphenethyl)-1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarboxamide(7q). Yield, 47%. ¹H NMR (CD₃OD) δ 7.52 (s, 2H), 7.27 (s, 2H), 7.23 (d,J=7.8 Hz, 4H), 7.15 (d, J=7.8 Hz, 4H), 4.02 (s, 6H), 3.92 (s, 6H), 3.91(m, 4H), 3.49 (s, 6H), 3.01 (t, J₁=J₂=6.6 Hz, 4H), 2.61 (q, J₁=J₂=7.8Hz, 6H), 2.11 (s, 6H), 1.21 (t, J₁=J₂=7.8 Hz, 6H).

N⁵,N⁵′-bis(2,3-dihydro-1H-inden-2-yl)-1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarboxamide(7s). Yield, 47%. ¹H NMR (CD₃OD) δ 7.50 (s, 2H), 7.45 (s, 2H), 7.26 (m,4H), 7.15 (m, 4H), 4.94 (m, 2H), 3.99 (s, 6H), 3.90 (s, 6H), 3.56 (s,6H), 3.43 (m, 4H), 3.07 (m, 4H), 2.08 (s, 6H).

1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-N⁵,N⁵′-diphenyl-2,2′-binaphthyl-5,5′-dicarboxamide(8a). Yield, 75%. ¹H NMR (CD₃OD) δ 7.77 (d, J=7.8 Hz, 4H), 7.63 (s, 2H),7.38 (t, J₁=7.8 Hz, J₂=7.2 Hz, 4H), 7.28 (s, 2H), 7.16 (t, J₁=7.8 Hz,J₂=7.2 Hz, 2H), 2.01 (s, 6H).

N⁵,N⁵′-dicyclopentyl-1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarboxamide(8b). Yield, 76%. ¹H NMR (CD₃OD) δ 7.57 (s, 2H), 7.19 (s, 2H), 4.46 (m,2H), 2.09 (m, 4H), 1.97 (s, 6H), 1.80 (m, 4H), 1.69 (m, 8H).

1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-N⁵,N⁵′-bis(4-phenoxyphenyl)-2,2′-binaphthyl-5,5′-dicarboxamide(8c). Yield, 65%. ¹H NMR (CD₃OD) δ 7.78 (d, J=8.4 Hz, 4H), 7.64 (s, 2H),7.35 (t, J₁=7.8 Hz, J₂=7.8 Hz, 4H), 7.28 (s, 2H), 7.08 (m, 2H), 7.02 (m,8H), 2.01 (s, 6H).

N⁵,N⁵′-bis(3-ethylphenyl)-1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarboxamide(8d). Yield, 69%. ¹H NMR (CD₃OD) δ7.63 (s, 4H), 7.60 (d, J=7.8 Hz, 2H),7.28 (m, 4H), 7.02 (d, J=7.8 Hz, 2H), 2.68 (q, J₁=J₂=8.4 Hz, 4H), 2.01(s, 6H), 1.28 (t, J₁=J₂=8.4 Hz, 6H).

1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-N⁵,N⁵′-bis(3-(trifluoromethyl)phenyl)-2,2′-binaphthyl-5,5′-dicarboxamide(8e). Yield, 69%. ¹H NMR (CD₃OD) δ 8.26 (s, 2H), 7.96 (d, J=7.8 Hz, 2H),7.65 (s, 2H), 7.57 (t, J₁=J₂=6.6 Hz, 2H), 7.44 (d, J=7.2 Hz, 2H), 7.27(s, 2H), 2.01 (s, 6H).

1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-N⁵,N⁵′-bis(1-phenylpropyl)-2,2′-binaphthyl-5,5′-dicarboxamide(8f). Yield, 70%. ¹H NMR (CD₃OD) δ 7.50 (m, 4H), 7.31 (m, 6H), 7.09 (s,2H), 6.95 (s, 2H), 5.09 (m, 2H), 1.88 (m, 6H), 1.09 (m, 6H).

N⁵,N⁵′-dibenzyl-1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarboxamide(8g). Yield, 78%. ¹H NMR (CD₃OD) δ 7.58 (s, 2H), 7.54 (d, J=7.2 Hz, 4H),7.36 (t, J₁=J₂=7.2 Hz, 4H), 7.27 (t, J₁=J₂=7.2 Hz, 2H), 7.16 (s, 2H),4.70 (s, 4H), 1.91 (s, 6H).

1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-N⁵,N⁵′-bis(3-methylbenzyl)-2,2′-binaphthyl-5,5′-dicarboxamide(8h). Yield, 75%. ¹H NMR (CD₃OD) δ 7.58 (s, 1H), 7.36 (s, 2H), 7.30 (d,J=7.8 Hz, 2H), 7.23 (t, J₁=7.8 Hz, J₂=7.2 Hz, 2H), 7.16 (s, 2H), 7.08(d, J=7.2 Hz, 2H), 4.65 (t, J₁=J₂=15.0 Hz, 4H), 2.36 (s, 6H), 1.91 (s,6H).

N⁵,N⁵′-bis(3-chlorobenzyl)-1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarboxamide(8i). Yield, 70%. ¹H NMR (CD₃OD) δ 7.59 (d, J=4.2 Hz, 4H), 7.46 (d,J=7.2 Hz, 2H), 7.35 (t, J₁=J₂=7.2 Hz, 2H), 7.28 (d, J=7.2 Hz, 2H), 7.15(s, 2H), 4.68 (s, 4H), 1.93 (s, 6H).

1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-N⁵,N⁵′-bis(2,4,6-trimethylbenzyl)-2,2′-binaphthyl-5,5′-dicarboxamide(8j). Yield, 70%. ¹H NMR (CD₃OD) δ 7.54 (s, 2H), 7.18 (s, 2H), 6.87 (s,4H), 4.70 (s, 4H), 2.46 (s, 12H), 2.22 (s, 6H), 1.91 (s, 6H).

N⁵,N⁵′-bis(1-(4-chlorophenyl)ethyl)-1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarboxamide(8k). Yield, 73%. ¹H NMR (CD₃OD) δ 7.53 (m, 6H), 7.35 (m, 4H), 7.09 (s,2H), 6.95 (s, 2H), 5.33 (m, 2H), 1.91 (s, 3H), 1.86 (s, 3H), 1.56 (m,3H), 1.54 (m, 3H).

N⁵,N⁵′-bis(cyclopropylmethyl)-1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarboxamide(8l). Yield, 70%. ¹H NMR (CD₃OD) δ 7.58 (s, 2H), 7.26 (s, 2H), 3.36 (m,4H), 1.97 (s, 6H), 1.18 (m, 2H), 0.57 (d, J=8.1 Hz, 4H), 0.37 (m, 4H).

N⁵,N⁵′-bis(cyclohexylmethyl)-1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarboxamide(8m). Yield, 80%. ¹H NMR (CD₃OD) δ 7.58 (s, 2H), 7.22 (s, 2H), 3.32 (m,4H), 1.96 (s, 6H), 1.79 (d, J=7.2 Hz, 4H), 1.71 (d, J=8.4 Hz, 4H),1.39-1.08 (m, 14H).

1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-N⁵,N⁵′-diphenethyl-2,2′-binaphthyl-5,5′-dicarboxamide(8n). Yield, 80%. ¹H NMR (CD₃OD) δ 7.58 (s, 2H), 7.36 (d, J=7.2 Hz, 4H),7.31 (t, J₁=J₂=7.2 Hz, 4H), 7.21 (t, J₁=J₂=7.2 Hz, 2H), 7.09 (s, 2H),3.74 (m, 4H), 3.01 (t, J₁=J₂=7.2 Hz, 4H), 1.92 (s, 6H).

1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-N⁵,N⁵′-bis(3-methylphenethyl)-2,2′-binaphthyl-5,5′-dicarboxamide(8o). Yield, 76%. ¹H NMR (CD₃OD) δ 7.57 (s, 2H), 7.23 (d, J=7.8 Hz, 4H),7.11 (d, J=7.8 Hz, 4H), 7.06 (s, 2H), 3.80 (m, 4H), 2.96 (t, J₁=J₂=7.2Hz, 4H), 2.29 (s, 6H), 1.90 (s, 6H), 1.40 (s, 4H).

N⁵,N⁵′-bis(3-chlorophenethyl)-1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarboxamide(8p). Yield, 70%. ¹H NMR (CD₃OD) δ 7.57 (s, 2H), 7.39 (s, 2H), 7.30 (t,J₁=7.2 Hz, J₂=6.6 Hz, 4H), 7.21 (d, J=6.6 Hz, 2H), 7.03 (s, 2H), 3.79(m, 2H), 3.70 (m, 2H), 3.00 (m, 4H), 1.91 (s, 6H).

N⁵,N⁵′-bis(4-ethylphenethyl)-1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarboxamide(8q). Yield, 75%. ¹H NMR (CD₃OD) δ 7.58 (s, 2H), 7.26 (d, J=7.8 Hz, 4H),7.15 (d, J=7.8 Hz, 4H), 7.10 (s, 2H), 3.75 (m, 2H), 3.70 (m, 2H), 2.98(t, J₁=J₂=7.2 Hz, 4H), 2.60 (q, J₁=7.8 Hz, J₂=7.2 Hz, 4H), 1.91 (s, 6H),1.20 (t, J₁=7.8 Hz, J₂=7.2 Hz, 6H).

N⁵,N⁵′-bis(2,3-dihydro-1H-inden-2-yl)-1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarboxamide(8s). Yield, 72%. ¹H NMR (CD₃OD) δ 7.57 (s, 2H), 7.24 (s, 4H), 7.19 (s,2H), 7.14 (s, 4H), 4.94 (m, 2H), 3.42 (m, 4H), 3.07 (m, 4H), 1.94 (s,6H).

N⁵,N⁵′-bis(4-chlorophenethyl)-1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarboxamide(8t). Yield, 75%. ¹H NMR (CD₃OD) δ 7.57 (s, 2H), 7.35 (d, J=7.8 Hz, 4H),7.30 (d, J=7.8 Hz, 4H), 7.02 (s, 2H), 3.76 (m, 2H), 3.71 (m, 2H), 2.99(t, J₁=J₂=6.6 Hz, 4H), 1.93 (s, 6H).

5,5′-diisobutyl-1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-2,2′-binaphthyl(11a). Yield, 85%. ¹H NMR (CDCl₃) δ 7.60 (s, 2H), 7.41 (s, 2H), 3.99 (s,6H), 3.90 (s, 6H), 3.57 (s, 6H), 2.97 (d, J=7.2 Hz, 4H), 2.19 (s, 6H),2.12 (m, 2H), 1.03 (t, J₁=J₂=6.0 Hz, 12H).

5,5′-diisopentyl-1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-2,2′-binaphthyl(11b). Yield, 81%. ¹H NMR (CDCl₃) δ 7.63 (s, 2H), 7.38 (s, 2H), 3.99 (s,6H), 3.96 (s, 6H), 3.59 (s, 6H), 3.08 (m, 4H), 2.2 (s, 6H), 1.80 (m,2H), 1.29 (m, 4H), 1.06 (m, 12H).

5,5′-bis(cyclopentylmethyl)-1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-2,2′-binaphthyl(11c). Yield, 80%. ¹H NMR (CDCl₃) δ 7.65 (s, 2H), 7.40 (s, 2H), 3.99 (s,6H), 3.90 (s, 6H), 3.58 (s, 6H), 3.09 (d, J=7.2 Hz, 4H), 2.38 (m, 2H),2.20 (s, 6H), 1.73 (m, 8H), 1.54 (m, 8H).

1,1′,6,6′,7,7′-hexamethoxy-3,3′-dimethyl-2,2′-binaphthyl (11e). Yield,90%. ¹H NMR (CDCl₃) δ 7.46 (s, 2H), 7.45(s, 2H), 7.14 (s, 2H), 4.04 (s,6H), 4.02 (s, 6H), 3.57 (s, 6H), 2.18 (s, 6H).

5,5′-diisobutyl-3,3′-dimethyl-2,2′-binaphthyl-1,1′,6,6′,7,7′-hexaol(12a). Yield, 80%. ¹H NMR (CD₃OD) δ 7.44 (s, 2H), 7.34 (s, 2H), 2.95 (d,J=7.2 Hz, 4H), 2.14 (m, 2H), 2.05 (s, 6H), 1.02 (d, J=6.0 Hz, 6H), 1.00(d, J=6.0 Hz, 6H).

5,5′-diisopentyl-3,3′-dimethyl-2,2′-binaphthyl-1,1′,6,6′,7,7′-hexaol(12b). Yield, 79%. ¹H NMR (CD₃OD) δ 7.42 (s, 2H), 7.34 (s, 2H), 3.04 (t,J₁=J₂=5.4 Hz, 4H), 2.05 (s, 6H), 1.74 (m, 2H), 1.55 (m, 4H), 1.05 (d,J=3.6 Hz, 6H), 1.04 (d, J=3.6 Hz, 6H).

5,5′-bis(cyclopentylmethyl)-3,3′-dimethyl-2,2′-binaphthyl-1,1′,6,6′,7,7′-hexaol(12c). Yield, 78%. ¹H NMR (CD₃OD) δ 7.41 (s, 2H), 7.37 (s, 2H), 3.06 (d,J=7.2 Hz, 4H), 2.36 (m, 2H), 2.03 (s, 6H), 1.72 (m, 8H), 1.50 (m, 8H).

5,5′-dibenzyl-3,3′-dimethyl-2,2′-binaphthyl-1,1′,6,6′,7,7′-hexaol (12d).Yield, 72%. ¹H NMR((CD₃)₂SO) δ 9.81 (s, 2H), 8.64 (s, 2H), 7.76 (s, 2H),7.39 (s, 2H), 7.24 (m, 10H), 7.10 (m, 2H), 4.28 (dd, J₁=15.0 Hz, J₂=19.8Hz, 4H), 1.94 (s, 6H). E1 ¹³C NMR ( ) δ 150.69, 145.86, 145.66, 143.20,134.04, 126.97, 120.46, 119.85, 117.38, 116.13, 105.10, 101.09, 32.0,22.5.

3,3′-dimethyl-5,5′-diphenethyl-2,2′-binaphthyl-1,1′,6,6′,7,7′-hexaol(12e). Yield, 73%. ¹H NMR (CDCl₃) δ 7.52 (s, 2H), 7.44 (s, 2H), 7.32 (d,J=6.6 Hz, 4H), 7.29 (d, J=7.2 Hz, 4H), 7.18 (t, J₁=7.2 Hz, J₁=6.6 Hz,2H), 5.35 (s, 4H), 5.17 (s, 2H), 3.37 (t, J₁=J₂=6.6 Hz, 4H), 3.03 (t,J₁=J₂=6.6 Hz, 4H), 2.13 (s, 6H).

3,3′-dimethyl-2,2′-binaphthyl-1,1′,6,6′,7,7′-hexaol (13). Yield, 75%. ¹HNMR (CD₃OD) δ 7.46 (s, 2H), 7.11 (s, 2H), 7.02 (s, 2H), 1.97 (s, 6H).

1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-2,2′-binaphthyl-5,5′-dicarboxylicacid (14). Yield, 70%. ¹H NMR (CD₃OD) δ 8.29 (s, 2H), 7.83 (s, 2H), 2.04(s, 6H).

TABLE 8 HIGH RESOLUTION MASS (HRMS) AND HPLC PURITY OF 5,5′-SUBSTITUTEDAPOGOSSYPOL DERIVATIVES HRMS [M + H]⁺ HPLC Compd. Chemical FormulaCalculated Found Purity (%) 2 C₂₈H₃₀O₆ 463.2115 463.2108 99.5(Apogossypol) 8a C₃₆H₂₈N₂O₈ 617.1918 617.1912 99.0 8b C₃₄H₃₆N₂O₈601.2544 601.2531 98.7 8g C₃₈H₃₂N₂O₈ 645.2231 645.2237 98.5 8cC₄₈H₃₆N₂O₁₀ 801.2443 801.2425 99.0 8n C₄₀H₃₆N₂O₈ 673.2544 673.2536 99.48d C₄₀H₃₆N₂O₈ 673.2544 673.2537 99.5 8e C₃₈H₂₆F₆N₂O₈ 753.1666 751.150698.5 8h C₄₀H₃₆N₂O₈ 673.2544 673.2536 97.5 8i C₃₈H₃₀N₂O₈ 713.1452713.1426 98.9 8j C₄₄H₄₄N₂O₈ 729.3170 729.3167 99.5 8l C₃₂H₃₂N₂O₈573.2231 573.2214 99.2 8k C₄₀H₃₄N₂O₈ 741.1765 741.1763 99.5 8mC₃₈H₄₄N₂O₈ 657.3170 657.3169 99.8 8p C₄₀H₃₄N₂O₈ 741.1765 741.1769 99.58q C₄₄H₄₄N₂O₈ 729.3170 729.3175 99.7 8r C₄₂H₄₀N₂O₈ 701.2857 701.286499.5 8s C₄₂H₃₆N₂O₈ 697.2544 697.2541 99.0 8t C₄₀H₃₄N₂O₈ 741.1765741.1765 98.0 8f C₄₀H₄₀N₂O₈ 701.2857 701.2867 99.0 8o C₄₀H₄₀N₂O₈701.2857 701.2859 99.0 12a C₃₀H₃₄O₆ 491.2428 491.2429 99.1 12b C₃₂H₃₈O₆519.2741 519.2739 99.5 12c C₃₄H₃₈O₆ 543.2741 543.2739 99.3 12d C₃₆H₃₀O₆559.2115 559.2112 99.5 12e C₃₈H₃₄O₆ 587.2428 587.2425 99.0 13 C₂₂H₁₈O₆379.1176 379.1168 98.5 14 C₂₄H₁₈O₁₀ 467.0973 467.0964 99.4

Example 6 NMR Experiments

NMR-based binding assays have been conducted by acquiringone-dimensional ¹H experiments with 500 μL solution of BCL-X_(L) at 25μM concentration, in absence and presence of added compounds, each at200 μM concentration. By observing the aliphatic region of the spectra,binding could be readily detected due to chemical shift changes inactive site methyl groups of Ile, Leu, Thr, Val or Ala (region between−0.8 and 0.3 ppm). All experiments were performed with a 600 MHzspectrometer Bruker Avance 600 equipped with four rf channels and z-axispulse-field gradients.

Example 7 Fluorescence Polarization Assays (FPA)

A Bak BH3 peptide (F-BakBH3) (GQVGRQLAIIGDDINR) was labeled at theN-terminus with fluorescein isothiocyanate (FITC) (Molecular Probes) andpurified by HPLC. For competitive binding assays, 100 nM GST-BCL-X_(L)ΔTM protein was preincubated with the tested compound at varyingconcentrations in 47.5 μL PBS (pH=7.4) in 96-well black plates at roomtemperature for 10 min, then 2.5 μL of 100 nM FITC-labeled Bak BH3peptide was added to produce a final volume of 50 μL. The wild-type andmutant Bak BH3 peptides were included in each assay plate as positiveand negative controls, respectively. After 30 min incubation at roomtemperature, the polarization values in millipolarization units weremeasured at excitation/emission wavelengths of 480/535 nm with amultilabel plate reader (PerkinElmer). IC₅₀ was determined by fittingthe experimental data to a sigmoidal dose-response nonlinear regressionmodel (SigmaPlot 10.0.1, Systat Software, Inc., San Jose, Calif., USA).Data reported are mean of three independent experiments±standard error(SE). Performance of BCL-2 and Mcl-1 FPA are similar. Briefly, 50 nM ofGST-BCL-2 or -Mcl-1 were incubated with various concentrations ofApogossypol, or its 5,5′ substituted derivatives for 2 min, then 15 nMFITC-conjugated-Bim BH3 peptide was added in PBS buffer. Fluorescencepolarization was measured after 10 min.

Example 8 Isothermal Titration Calorimetry Assays (ITC)

Titrations were performed using a VP-ITC or ITC200 calorimeter fromMicrocal (Northampton, Mass.). BCL-X_(L) was used at concentrationsbetween 25 and 100 μM in 20 mM sodium phosphate buffer (pH 7.4) and5-10% DMSO. Titrants were used at concentrations 10-15 fold of that ofthe protein in the same buffer. Titrations were carried out at 25° C.Data were analyzed using Microcal Origin software provided by the ITCmanufacturer (Microcal, Northampton, Mass.).

Example 9 Cell Viability and Apoptosis Assays

The activity of the compounds against human cancer cell lines (PC3ML,H460, H1299, RS11846) were assessed by using the ATP-LITE assay(PerkinElmer). All cells were seeded in either F12 or RPMI1640 mediumwith 5 mM L-glutamine supplemented with 5% fetal bovine serum (MediatechInc.), penicillin and streptomycin (Omega). For maintenance, cells werecultured in 5% FBS. Cells plated into 96 well plates at varying initialdensities depending on doubling time. H460 and H1299 plated at 2000cells/well, A549 and PC3 at 3000 cells/well, and RS118456S at 10,000cells/well. Compounds were diluted to final concentrations with 0.1%DMSO. Prior to dispensing compounds onto cells, fresh 5% media wasplaced into wells. Administration of compounds occurred 24 hours afterseeding into the fresh media. Cell viability was evaluated usingATP-LITE reagent (PerkinElmer) after 72 hours of treatment. Data werenormalized to the DMSO control-treated cells using Prism version 5.01(Graphpad Software).

The apoptotic activity of the compounds against RS11846 cells wasassessed by staining with Annexin V- and propidium iodide (PI). Lymphomacell line, RS11846, was cultured in RPMI 1640 medium (Mediatech Inc.,Herndon, Va. 20171) containing 10% fetal bovine serum (Mediatech Inc.,Herndon, Va. 20171) and Penicillin/Streptomycin (Mediatech Inc.,Herndon, Va. 20171). Cells were cultured with various concentrations of5,5′ substituted Apogossypol for 1-2 days. The percentage of viablecells was determined by FITC-Annexin V- and propidium iodide(PI)-labeling, using an Apoptosis Detection kit (BioVision Inc.), andanalyzing stained cells by flow cytometry (FACSort; Bectin-Dickinson,Inc.; Mountain View, Calif.). Cells that were annexin-V-negative andPI-negative were considered viable.

The apoptotic activity of the compounds, such as 8r, 8q against mouseembryonic fibroblast wild-type cells (MEF/WT) and mouse embryonicfibroblast BAX/Bak double knockout cells (MEF/DKO) was assessed bystaining with Annexin V- and propidium iodide (PI). MEF/WT and MEF/DKOcells were seeded in 24-well plate at a seeding density of half amillion per well (in 1 ml of DMEM medium supplemented by 10% FCS). Nextday, compound was added to wild-type and DKO cells at finalconcentration of 0, 2.5, 5.0, 7.5 and 10 μM. On the following day,floating cells were pooled with adherent cells harvested after briefincubation with 0.25% Trypsin/EDTA solution (Gibco/In-Vitrogen Inc.).Cells were centrifuged and supernatant was discarded, and cell pelletwas re-suspended with 0.2 ml of Annexin-V binding buffer, followed byaddition of 1 μl Annexin-FITC and 1 μl PI (propidium iodide). Thepercentage of viable cells was determined by a 3-color FACSortinstrument and data analyzed by Flow-Jo program, scoring AnnexinV-negative, PI-negative as viable cells.

Example 10 In vitro Admet Studies

Liver Microsomal Stability. Pooled rat liver microsomes (BD Biosciences,#452701) were preincubated with test compounds at 37.5° C. for 5 min inthe absence of NADPH. The reaction was initiated by addition of NADPHand then incubated under the same conditions. The final incubationconcentrations were 4 μM test compound, 2 mM NADPH, and 1 mg/mL (totalprotein) liver microsomes in phosphate-buffered saline (PBS) at pH 7.4.One aliquot (100 μL) of the incubation mixture was withdrawn at 0, 15,30, and 60 min and combined immediately with 200 μL of ACN/MeOHcontaining an internal standard. After mixing, the sample wascentrifuged at approximately 13,000 rpm for 12 min. The supernatant wastransferred into an autosampler vial and the amount of test compound wasquantified using the Shimadzu LCMS 2010EV mass spectrometer. The changeof the AUC (area under the curve) of the parent compound as function oftime was used as a measure of microsomal stability.

Plasma Stability. A 20 μL aliquot of a 10 mM solution in DMSO of thetest compound was added to 2.0 mL of heparinized rat plasma (Lampire,P1-150N) to obtain a 100 μM final solution. The mixture was incubatedfor 1 h at 37.5° C. Aliquots of 100 μL were taken (0, 30 min, 1 h) anddiluted with 200 μL of MeOH containing internal standard. After mixing,the sample was centrifuged at approximately 13,000 rpm for 12 min. Thesupernatant was transferred into an autosampler vial and the amount oftest compound was quantified using the Shimadzu LCMS-2010EV system. Thechange of the AUC (area under the curve) of the parent compound asfunction of time was used as a measure of microsomal stability.

Example 11 PAMPA Assays

PAMPA is parallel artificial membrane permeation assay. A 96-wellmicrotiter plate (Millipore, #MSSACCEPTOR) was completely filled withaqueous buffer solution (pH 7.2) and covered with a microtiterfilterplate (Millipore, #MAPBMN310). The hydrophobic filter material wasimpregnated with a 10% solution of hexadecane in hexane and the organicsolvent was allowed to completely evaporate. Permeation studies werestarted by the transfer of 200 μL of a 100 μM test compound solution ontop of the filterplate. In general phosphate buffer at pH 7.2 buffer wasused. The maximum DMSO content of the stock solutions was <5%. Inparallel, an equilibrium solution lacking a membrane was prepared usingthe exact concentrations and specifications but lacking the membrane.The concentrations of the acceptor and equilibrium solutions weredetermined using the Shimadzu LCMS-2010EV and AUC methods. Thepermeation of a compound through the membrane layer is described by thepercentage permeation (% flux). The flux values were calculatedconsidering the concentration of the acceptor compartment after 8 h andthat of a reference well with the same concentration containing nomembrane bather.

Example 12 Transgenic Mice Studies

Transgenic mice expressing BCL-2 have been described as the B6 line. TheBCL-2 transgene res a minigene version of a t(14;18) translocation inwhich the human BCL-2 gene is fused with the immunoglobulin heavy-chain(IgH) locus and associated IgH enhancer. The transgene was propagated onthe Balb/c background. These mice develop polyclonal B-cell hyperplasiawith asynchronous transformation to monoclonal aggressive lymphomasbeginning at approximately 6 months of age, with approximately 90% ofmice undergoing transformation by the age of 12 to 24 months. Allanimals used here had not yet developed aggressive lymphoma.

Example 13 Further Mouse Experiments

Compounds dissolved in 500 μL of solution (Ethanol:CremophorEL:Saline=10:10:80) were injected intraperitoneally to age- andsex-matched B6BCL2 mouse, while control-mice were injectedintraperitoneally with 500 μL of the same formulation without compound.After 24 hours, B6BCL2 mice were sacrificed by intraperitoneal injectionof lethal dose of Avertin. Spleen was removed and weighed. The spleenweight of mice is used as an end-point for assessing activity as wedetermined that spleen weight is highly consistent in age- andsex-matched BCL-2-transgenic mice in preliminary studies. Variability ofspleen weight was within ±2% among control-treated age-matched,sex-matched B6BCL2 mice. Spleen tissue was fixed in z-FIX for 3 days andrinsed in PBS, and saved for histological analysis of spleen (H&Estaining and TUNEL assay).

Example 14 Comparisons with Apogossypol

Molecular docking studies of apogossypol into the BH3 binding groove inBCL-X_(L) suggest that apogossypol forms two hydrogen bonds withresidues Arg 139 and Tyr 195 in BCL-X_(L) through adjacent sixth andseventh hydroxyl groups on the right naphthalene ring. The isopropylgroup on the left naphthalene ring inserts into the first hydrophobicpocket (P1) in BCL-X_(L), while the methyl group and the isopropyl groupon the right naphthalene ring insert into the adjacent two hydrophobicpockets, P2 and P3, respectively. Analysis of the predicted bindingmodels indicates that while the overall core structure of apogossypolfits rather well into BH3 binding groove of BCL-X_(L), the two isopropylgroups do not apparently fully occupy the hydrophobic pockets P1 and P3.

Therefore, a library of 5,5′ substituted apogossypol derivatives thatreplace the isopropyl groups with larger hydrophobic substituents wasdesigned with the aim of deriving novel molecules that could occupy thehydrophobic pockets on BCL-X_(L) more efficiently.

The designed 5,5′ substituted apogossypol derivatives were synthesizedas described herein and evaluated by nuclear magnetic resonancespectroscopy (NMR) binding assays, competitive fluorescence polarizationassays (FPA), and cell viability assays as shown in Table 9.

TABLE 9 EVALUATION OF 5,5′ SUBSTITUTED APOGOSSYPOL DERIVATIVES USING ACOMBINATION OF 1D 1H NMR BINDING ASSAYS, COMPETITIVE FLUORESCENCEPOLARIZATION ASSAYS AND CELL VIABILITY ASSAYS

1D ¹H- FPA NMR IC₅₀ Binding (μM) PC3ML H460 H1299 Assaya (BCL- EC₅₀ EC₅₀EC₅₀ RS11846b RS11846c Compound R (BCL-XL) XL) (μM) (μM) (μM) EC₅₀ (μM)EC₅₀ (μM) Gossypol

++ 2.72 3.1 3.0 6.0 2.2 4.23 Apogossypol

++ 3.69 10.3 2.8 3.4 5.0 8.6 I

+++ 0.19 4.6 0.68 3.5 2.6 4.9 II —H + NR 12.6 10.1 13.4 10.0 24.7 III

++ NR 3.9 1.5 4.8 15 14.7 IV

+ 1.30 7.5 1.1 3.6 10 13.7 V

+ 1.29 3.0 1.5 3.0 2.8 6.6 VI

+ 0.45 3.4 1.1 3.1 4.0 4.5 VII

+ 2.9 3.6 0.31 4.2 NR 18.3 VIII

+ 0.16 3.0 0.59 2.4 1.8 4.2 IX

+ NR 7.7 8.2 9.6 2.8 25.9 X

− NR 2.8 3.6 4.8 2.3 13.4 XI

+ 0.25 2.9 2.2 2.0 2.5 3.8 XII

++ 0.32 2.5 0.82 1.7 2.2 3.0 XIII

++ 1.31 3.1 2.7 2.6 8.4 5.3 XIV

++ 1.30 1.9 3.3 3.9 1.8 6.2 XV

+ NR 1.9 1.8 2.1 2 5.2 XVI

++ 0.14 2.8 1.5 2.2 2.3 3.1 XVII

+ 0.39 5.2 1.4 5.8 2.9 7 XVIII

++ NR NR NR NR NR 14.7 XIX

+ NR NR NR NR NR 17.1 XX

+ NR NR NR NR NR 11.7 a4-point-rating scale: +++: Very Active; ++:Active; +: Mild; −: Weak bCompounds against RS11846 cell line usingATP-LITE assay cCompounds against RS11846 cell line using Annexin V-andpropidium iodide assay

Compound I displayed high affinity for BCL-X_(L) in these assays. Itinduced significant chemical shift changes in active site methyl groups(region between −0.3 and 0.8 ppm) in the one-dimensional ¹H-NMR spectraof BCL-X_(L) and also has an IC₅₀ value of 0.19 μM in the FPdisplacement assays, which is almost 20 times more effective thanapogossypol.

A group of compounds, such as compounds XVII, VI, VIII, XI, XVI, and XIIalso displayed high binding affinity to BCL-X_(L) in the FP assays withIC₅₀ values ranging from 0.14 to 0.45 μM and induced chemical shiftchanges in the one-dimensional ¹H-NMR spectra of BCL-X_(L). To confirmresults of the NMR binding data and the FP assays, the binding affinityof compound I and other compounds was further evaluated for BCL-X_(L)using ITC (Isothermal Titration Calorimetry).

TABLE 10 CROSS-ACTIVITY OF SELECTED 5,5′ SUBSTITUTED APOGOSSYPOLDERIVATIVES AGAINST BCL-XL, BCL-2, AND MCL-1

EC50 (uM) FPA Kd (μM) ITC Compound R BCL-XL BCL-2 Mcl-1 BCL-XLApogossypol

3.69 2.80 2.60 1.70 I

0.19 0.36 0.52 0.17 XII

0.32 0.78 1.10 0.04 VIII

0.16 1.90 2.20 2.75

As can be seen, in agreement with NMR binding and FPA data, compound Iand its para-methyl substituted derivative compound XII, displayedpotent binding affinity to BCL-X_(L) with K_(d) values of 0.17 and 0.04μM, respectively, which is 10 and 40 times more potent than apogossypol(K_(d)=1.7 μM) in the same assay. Molecular docking studies of compoundI in the BH3 binding groove of BCL-X_(L) demonstrated that 5,5′ benzylgroups insert deeper into hydrophobic pockets (P1 and P3) in BCL-X_(L)hence occupying these regions more efficiently compared to isopropylgroups of apogossypol.

Consistent with NMR binding, FPA, and ITC data, compounds such ascompounds I and XII display significant efficacy in inhibiting cellgrowth in PC3ML cells, which express high levels of BCL-X_(L). TheirEC₅₀ values ranged from 1.9 to 4.6 μM, hence 2-5 fold more potent thanapogossypol (EC₅₀=10.3 μM).

To evaluate the binding properties and specificity of 5,5′ substitutedapogossypol derivatives to other anti-apoptotic BCL-2 family proteins,selected BCL-X_(L) active compounds were evaluated against BCL-2 andMcl-1 using FP assays. These BCL-X_(L) inhibitors also displayed strongbinding affinity to BCL-2 and Mcl-1. Compound I binds to BCL-2 and Mcl-1with EC₅₀ values of 0.36 and 0.52 μM, respectively, which areapproximately 8 and 5 fold more potent than apogossypol (EC₅₀=2.8 μM).Compound XII is slightly less active than compound I, while compoundVIII has activity that is similar to that of apogossypol.

Since compounds I and XII displayed strong binding affinities to BCL-2and Mcl-1 in FP assay, all 5,5′ substituted apogossypol derivatives werefurther evaluated against H460 and H1299 cell lines, which express highlevels of BCL-2 and Mcl-1, respectively. In agreement with FPA data,compounds I and XII inhibited growth of the H460 cell line with EC₅₀values of 0.68 and 0.82 μM, respectively, which are approximately 4-5times more potent than apogossypol (EC₅₀=3.4 μM). Compounds VII and VIIIhaving structures that are similar to that of compound I also inhibitedcell growth in the H460 cell line with EC₅₀ values of 0.30 and 0.59 μM,respectively. Most of the tested 5,5′ substituted apogossypolderivatives also showed potent cell activity in the H460 and H1299 celllines with EC₅₀ values ranging from 1 to 4 μM.

In contrast, compound II, the negative control compound with hydrogenatoms on 5,5′ positions, displayed weak cell growth inhibition activityin both H460 (EC₅₀=10.1 μM) and H1299 (EC₅₀=13.4 μM) cell linesindicating 5,5′ substituted groups are necessary for strong inhibition.This observation is in agreement with reports for the potent BCL-X_(L)antagonist ABT-737, which is not effective against Mcl-1 andconsequentially is not effective in killing Mcl-1 overexpressing celllines such as the H1299.

5,5′ substituted apogossypol derivatives were further tested for theirability to induce apoptosis of the human lymphoma RS11846 cell line,which expresses high levels of BCL-2 and BCL-X_(L). For these assays, weused Annexin V-FITC and propidium iodide (PI) double staining, followedby flow-cytometry analysis. Most of synthesized apogossypol derivativeseffectively induced apoptosis of the RS11846 cell line in adose-dependent manner. In particular, compounds I, VIII, XI, and XIIhave EC₅₀ values ranging from 3.0 to 5.5 μM, which is consistent withprevious results in human cancer PC3ML and H460 cell lines. Again, thenegative control compound II induced weak apoptosis (EC₅₀=24.7) of theRS11846 cell line, consistent with its poor anti-BCL-2 activity.

To test the pharmacological properties of 5,5′ substituted apogossypolderivatives, their in vitro plasma stability, microsomal stability, andcell membrane permeability were determined. The results are shown inTable 11.

TABLE 11 PLASMA STABILITY, MICROSOMAL STABILITY, AND CELL PERMEABILITYOF SELECTED 5,5′ SUBSTITUTED APOGOSSYPOL DERIVATIVES Plasma MicrosomalStability Stability Cell Compound R (T = 1 hr) (T = 1 hr) PermeabilityApogossypol

53% 60% Low XVII

90% 68% Medium VI

79% 27% Low VIII

62% 52% Low I

85% 64% Medium XII

NR 41% Low XII

72% 92% Medium XVIII

90% 30% Medium

As can be seen from the data provided in Table 4, the synthesizedcompounds of the disclosure displayed superior plasma stability andoverall are more stable than apogossypol. Compounds I degraded 15% after1 hour incubation in rat plasma. In addition, compounds I and XII showedsimilar or improved microsomal stability compared to Apogossypol, whilecompounds VI and XVIII, degraded faster than apogossypol in rathepatocytes microsomal preparations. Compounds I and XII also displayedimproved cell membrane permeability compared to apogossypol.

Accordingly, using a combination of 1D ¹H-NMR binding assays, FP assays,ITC assays, cytotoxicity assays and preliminary in vitro ADME data,compounds such as compounds I and XII were selected for further in vivostudies using B6BCL-2 transgenic mice. B-cells of the B6BCL-2 transgenicmice overexpress BCL-2 and accumulate in the spleen of mice. The spleenweight is used as an end-point for assessing in vivo activity as wedetermined that the spleen weight is highly consistent in age- andsex-matched BCL-2-transgenic mice and variability was within ±2% amongcontrol-treated age-matched, sex-matched B6BCL2 mice. The in vivoactivities of compounds such as compounds I and XII were first screenedside by side with apogossypol and gossypol in a single BCL-2 transgenicmouse at 60 μmmol/kg.

All tested compounds induced significant spleen weight reduction of miceand compound I displayed best efficiency causing 40% reduction in spleenweight. Since the maximum spleen shrinkage would be no more than 50% inthis experimental model, the in vivo effect of compound I induced nearmaximal biological activity at 60 μmmol/kg. To confirm the result from asingle mouse experiment, the in vivo activity of compound I was nextevaluated in groups of six mice each. In agreement with the single mouseexperiment, compound I treatment of these mice resulted in a significant(˜40%) reduction of spleen weight (P<0.0001), compared to the controlgroup of six mice. All mice tolerated the treatment well with nomacroscopic toxicity; the maximal weight loss was 4% during the courseof study of compound I.

Example 15 Mouse Model for Prevention and Treatment of Systemic LupusErythematosus (SLE)

This example illustrates a proposed study to examine the effect ofapogossypol treatment on development of SLE in the New Zealand black×NewZealand white F1 (NZBW) and MRL/lpr mouse models.

Prevention Studies

Two genetically diverse strains, NZB/NZW F1 (which is geneticallysimilar to B6.Sle1.Sle3 congenics) and MRL/lpr would be subjected topreventative studies from the age of 3 mo, to the age of 5 mo, i.e., fora 2 month period. Mice will be checked to ensure they are negative foranti-nuclear autoantibodies at the beginning of the study. In oneexample, 10 mice of each strain will receive apogossypol, whereasanother 10 age/gender matched females will receive the vehicle (“placebogroup”). Although 10 mice will be tested initially, these numbers couldeasily be ramped up following the power analysis conducted using theinitial set of data obtained. Mice will be given from between about 0.2mol/kg to about 1.0 μmol/kg per day. The route of administration will beoral. However, intravenous administration can also be used.

The mice will be monitored at fortnightly intervals for serumautoantibody levels and 24-hour urine protein levels, and at monthlyintervals for full blood counts, numbers and activation status of bloodleukocytes (using flow cytometry). At the end of the study, all micewill also be examined for creatinine/BUN levels, spleen leukocyte countsand activation status, as well as histological severity of glomerularand interstitial lesions in their kidneys. Statistical analyses will becarried out to determine if the apogossypol treated mice havesignificantly reduced autoantibodies and leukocyte numbers/activation(primary outcome measures), or renal disease (secondary outcomemeasure). Finally, flow-sorted leukocyte populations from both studygroups will be examined for the phosphorylation status of BCL-2,BCL-x_(L), AKT, mTOR, Erk1,2, p38, CDK1/2, and NFkB, to ascertain ifBCL-2 blockade also dampens other hyperactivated signaling pathways inlupus.

Treatment Studies

The same two genetically diverse strains, NZB/NZW F1 and MRL/lpr wouldbe subjected to treatment studies from the age of 5 mo (once they arepositive for anti-nuclear autoantibodies and become proteinuric), to theage of 7 months, i.e., for a 2 month period. 20 mice of each strain willreceive apogossypol, whereas another 20 age/gender matched females willreceive the vehicle (“placebo group”). Mice will be given from betweenabout 0.2 μmol/kg to about 1.0 μmol/kg per day. The route ofadministration may be oral. However, intravenous administration can alsobe used. All mice will be tested to ensure they are positive foranti-nuclear autoantibodies at the beginning of the study. In oneexample, 10 mice will be sacrificed immediately after the treatmentperiod, to examine for splenic leukocyte numbers/activation, whereas theremaining 10 mice in each group will be followed up till death (in orderto ascertain the impact of apogossypol on mortality).

The mice will be monitored at fortnightly intervals for serumautoantibody levels and 24-hour urine protein levels, and at monthlyintervals for full blood counts, numbers and activation status of bloodleukocytes (using flow cytometry). At the end of the study, all micewill also be examined for creatinine/BUN levels, spleen leukocyte countsand activation status, as well as histological severity of glomerularand interstitial lesions in their kidneys. Statistical analyses will becarried out to determine if the apogossypol treated mice havesignificantly reduced autoantibodies, mortality and leukocytenumbers/activation (primary outcome measures), or renal disease(secondary outcome measure). Finally, flow-sorted leukocyte populationsfrom both study groups will be examined for the phosphorylation statusof BCL-2, BCL-x_(L), AKT, mTOR, Erk1,2, p38, CDK1/2, and NFkB, toascertain if BCL-2 blockade also dampens other hyperactivated signalingpathways in lupus.

Follow up studies will include: assessing the impact of BCL-2 blockadeon selected lupus checkpoints, assessing whether the combined use ofapogossypol and other conventional drugs might yield better therapeuticefficacy with reduced side-effects, assessing the level of generalizedimmunosuppression due to apogossypol, and assessing the level of BCL-2family member activation in human lupus.

Example 16 Prevention of Experimental Autoimmune Encephalomyelitis (EAE)in the Murine Model of Multiple Sclerosis

This example illustrates a proposed study to examine the effect ofapogossypol treatment on development of both active and passive EAE inthe murine model of multiple sclerosis.

Experimental allergic encephalomyelitis (EAE) is a T cell mediatedautoimmune disease of the central nervous system (CNS). Disease can beinduced in susceptible strains of mice by immunization with CNS myelinantigens or alternatively, disease can be passively transferred tosusceptible mice, such as SJL/J mice, using antigen stimulated CD4+Tcells (Pettinelli, J. Immunol. 127, 1981, p. 1420). EAE is widelyrecognized as an acceptable animal model for multiple sclerosis inprimates (Alvord et al. (eds.) 1984. Experimental allergicencephalomyelitis—A useful model for multiple sclerosis. Alan R. Liss,New York).

Prevention Studies

Female SJL/J mice would be subjected to preventative studies from theage of 7 to 10 weeks, i.e., for a 2 month period. In one example, 10mice will receive apogossypol, whereas another 10 age/gender matchedfemales will receive the vehicle (“placebo group”). Although 10 micewill be tested initially, these numbers could easily be ramped upfollowing the power analysis conducted using the initial set of dataobtained. Mice will be given from between about 0.2 μmol/kg to about 1.0μmol/kg per day. The route of administration will be oral. However,intravenous administration can also be used.

a) Active EAE. Active EAE would be induced by immunization of femaleSJL/J mice with, for example, about 800 μg of mouse spinal cordhomogenate (“MSCH”) in complete Freund's adjuvant (“CFA”) on days zeroand seven; following the procedure described in Racke et al., J.Neuroimmunol., Vol 46:175-184, (1993).

b) Passive EAE. Passive EAE would be induced by adoptive transfer ofmyelin basic protein (“MBP”)-sensitized T lymphocytes as follows: femaleSJL/J mice (four- to six-weeks-old) were immunized on days zero andseven with 400 μg of MBP in CFA. On day 14 the regional draining lymphnode cells and spleen are harvested and cultured. The cells are culturedat about 4×10⁶ cells/well in, for example, RPMI 1640 (Gibco,Gaithersburg, Md.) containing 10% fetal bovine serum (Hyclone Labs,Logan, Utah), 2 mM L-glutamine (Gibco, Gaithersburg, Md.), 5×10⁻⁵ M2-mercaptoethanol (Gibco, Gaithersburg, Md.), 1% penicillin/streptomycin(Gibco, Gaithersburg, Md.), and 100 μg/ml of MBP. After four days,viable T cell blasts are harvested, washed, and injectedintraperitoneally into recipient mice (1×10⁷ to 1.5×10⁷ cells in 500 μlof PBS).

The mice will be monitored at daily intervals for clinical signs of EAEand scored on a scale of 0 to 3 as follows: 0.5—Distal limp tail;1.0—Complete limp tail; 1.5—Limp tail and hind limb weakness (unsteadygait); 2.0—Partial hind limb paralysis; 3.0—Complete bilateral hind limbparalysis. At the end of the study, all mice will also be examined forlymphocyte infiltration and demyelination of the spinal cord.Statistical analyses will be carried out to determine if the apogossypoltreated mice have significantly reduced disease severity, inflammationand/or demyelination.

Example 17 Prevention and Treatment of Diabetes in the NOD/SCID MouseModel

This example illustrates generally the proposed use of the NOD/SCIDmouse model to test the ability of apogossypol to prevent or treatdiabetes.

The non-obese diabetic (NOD) mouse is a model for auto-immune disease,in this case insulin-dependent diabetes mellitus (IDDM), which mainclinical feature is elevated blood glucose levels (hyperglycemia). Theelevated blood glucose levels are caused by the immune-mediateddestruction of insulin-producing β cells in the islets of Langerhans ofthe pancreas. This destruction is accompanied by a massive cellularinfiltration surrounding and penetrating of the islets (insulitis) by aheterogeneous mixture composed of a CD4+ and CD8+T lymphocytes, Blymphocytes, macrophages and dendritic cells.

The NOD mouse model for inflammation was generally described previously.Female NOD mice spontaneously develop an IDDM-like disease withdestruction of the β cells in the pancreas and spilling of glucose intothe urine beginning around 12-14 weeks of age. A typical longitudinalhistological examination of the NOD pancreas demonstrates infiltratingcells surrounding the blood vessels at 3-4 weeks of age, but the isletsare typically still clear at 6-7 weeks. Infiltrating cells than reachthe islets, either surrounding them or accumulating at one pole. Between10 and 12 weeks, the infiltrating cells penetrate into the islets andthe islets become swollen with lymphocytes. The easiest and mostreliable way to detect the onset of diabetes in these mice is to testfor glucose levels in the blood.

Diabetes can be assessed by measurement of venous blood using, forexample, an Abbott Medisense Precision Q.I.D. glucometer and alsomonitored for glucosuria (Gluketur Test; Boehringer Mannheim, Mannheim,Germany). Animals will be considered diabetic after two consecutiveglucose measurements of higher than about 13.75 mmol/l (250 mg/dl).Onset of diabetes will be dated from the first consecutive reading. Ininstances of sustained hyperglycemia of >33 mmol/l animals will besacrificed to avoid prolonged discomfort.

Prevention Studies

NOD/LtJ mice (Jackson Laboratories) would be subjected to preventativestudies from the age of about 8-10 weeks, for a 2 month period. Micewill be checked to ensure they are negative for IDDM-like diseasesymptoms at the beginning of the study. In one example, 10 will receiveapogossypol, whereas another 10 age/gender matched females will receivethe vehicle (“placebo group”). Although 10 mice will be testedinitially, these numbers could easily be ramped up following the poweranalysis conducted using the initial set of data obtained. Mice will begiven from between about 0.2 μmol/kg to about 1.0 μmol/kg per day. Theroute of administration may be oral; intravenous administration can alsobe used.

The mice will be monitored at daily intervals for blood glucose levelsand 24-hour urine protein and glucose levels, and at monthly intervalsfor full blood counts, numbers and activation status of blood leukocytes(using flow cytometry). At the end of the study, all mice will also beexamined for insulin levels, presence of CD4+ and CD8+T lymphocytes, Blymphocytes, macrophages and dendritic cells in the pancreas, as well asgeneral morphology of the pancreas. Statistical analyses will be carriedout to determine if the apogossypol treated mice have significantlydelayed onset of diabetes.

Treatment Studies

NOD/LtJ mice would be subjected to treatment studies beginning at about10-12 weeks of age. 20 mice will receive apogossypol, whereas another 20age/gender matched females will receive the vehicle (“placebo group”).Mice will be given from between about 0.2 μmol/kg to about 1.0 μmol/kgper day. The route of administration will be oral; intravenousadministration can also be used. All mice will be tested to ensure theyare positive for IDDM-like disease (i.e., two consecutive glucosemeasurements of higher than about 13.75 mmol/l (250 mg/dl)) at thebeginning of the study. In one example, 10 mice will be sacrificedimmediately after the treatment period, to examine for pancreasmorphology and presence of lymphocytes in the pancreas, whereas theremaining 10 mice in each group will be followed up till death (in orderto ascertain the impact of apogossypol on mortality).

The mice will be monitored at daily intervals for blood glucose levelsand 24-hour urine protein and glucose levels, and at monthly intervalsfor full blood counts, numbers and activation status of blood leukocytes(using flow cytometry). At the end of the study, all mice will also beexamined for insulin levels, presence of CD4+ and CD8+T lymphocytes, Blymphocytes, macrophages and dendritic cells in the pancreas, as well asgeneral morphology of the pancreas. Statistical analyses will be carriedout to determine if the apogossypol treated mice have significantlyreduced blood glucose levels, urine glucose levels, and mortality andleukocyte numbers/activation.

Follow up studies will include: assessing the impact of BCL2 blockade onselected IDDM-like disease checkpoints, assessing whether the combineduse of apogossypol and other conventional drugs might yield bettertherapeutic efficacy with reduced side-effects, assessing the level ofgeneralized immunosuppression due to apogossypol, and assessing thelevel of BCL2 family member activation in human diabetes.

Example 18 Studies of Apogossypol Activity and Toxicity in BCL-2Transgenic Mice

The toxicity and efficacy studies were conducted in mice to comparegossypol and apogossypol. At daily dose of 0.12 mmol/kg p.o., %mortality in gossypol-treated Balb/c mice was 100% by the end of week 3.Gossypol-treated mice developed the following toxicities: GI toxicity(partial paralytic ileus), hematological toxicity (lymphopenia),hepatotoxicity (elevation of serum levels of ALT and AST), weight lossand cardic toxicity, and cause of death was cardiac failure ingossypol-treated mice.

FIGS. 5A and 5B further illustrate toxicity profiles of gossypol vs.apogossypol. FIG. 5A shows % survival in young, healthy Balb/c mice(7-weeks-old females, 6 mice per group). Mice were orally administeredwith apogossypol, gossypol or vehicle-control at a daily dose of 0.12mmol/kg, QDx5 for three weeks. % survival dropped to 0 by the end of 3weeks of treatment with gossypol, whereas % survival remained high amonggroups treated with apogossypol or vehicle-control.

FIG. 5B illustrates changes in body weight, which were monitoredthroughout the entire period of treatments with apogossypol, gossypol orvehicle-control at a daily dose of 0.12 mmol/kg, QDx5 for three weeks.Data expressed in grams (Mean±Standard Deviation).

As can be seen from the data provided by FIGS. 5A and 5B, apogossypolwas less toxic than gossypol in all these categories and apogossypol didnot induce any abnormal changes in E.C.G. pattern throughout the entireperiod of treatment. Gossypol-treated mice became lethagic with scruffyhair, whereas apogossypol or vehicle-control-treated mice remainedactive and apparently healthy without weight loss throughout thetreatment period. Apogossypol-treated mice as well as vehicle-controlmice (0.12 mmol/kg ascorbic acid in sesame oil) revealed normalE.C.G.-pattern, using 2-electrodes, by the use of MP150 Biopac system(the third electrode=ground). In addition, apogossypol-treated mice aswell as vehicle-control mice exhibited normal bowel movement inultrasound imaging-Cinema (300 frames), while no weight loss was notedduring the entire course of treatment. One of 6 apogossypol-treated micewas found dead on day 18 of treatment. This mouse was apparently healthyuntil the day before death, and cause of death is unknown at thismoment.

Apogossypol was well-tolerated in nude mice grafted with SCLC H146 cellline at daily dose of 0.24 mmol/kg, p.o. (no fatality and no weightloss), and anti-tumor effect of apogossypol was demonstrated. Ascorbicacid-stabilized apogossypol was stable for 2.5 weeks when stored at 4°C. or at room temperature under nitrogen gas or air, with or withoutlight.

Toxicity

It was determined that apogossypol is less toxic than gossypol. Thetoxicities of gossypol and apogossypol were compared in normal femaleBalb/c mice. Preliminary maximum tolerated dose (MTD) studies suggestedthat apogossypol was less toxic than gossypol whether delivered orallyor by intraperitoneal injection. Previous NCI-sponsored studiesdetermined that racemic gossypol and (−)gossypol are non-lethal and showanti-tumor activity when dosed orally at 0.06 mmol/kg daily for up to 21days. Thus, orally administered gossypol and apogossypol were comparedat twice this dose; animals were dosed with 0.12 mmol/kg. Ascorbic acidwas employed as a control, because apogossypol is formulated at 1:1molar ratio with this weak acid, which renders the compound stable uponstorage. Compounds or vehicle control were dosed 15 times over 3 weeks,giving compounds daily for 5 consecutive days (Monday-Friday), restingon weekends.

BCL-2 Transgenic Mice

Transgenic mice expressing BCL-2 have been described as the B6 line. TheBCL-2 transgene res a mini-gene version of a t(14;18) translocation inwhich the human BCL-2 gene is fused with the immunoglobulin heavy-chain(IgH) locus and associated IgH enhancer. The transgene was propagated onthe Balb/c background.

Patient Specimens

Peripheral blood mononuclear cells (PBMC) from patients with CLL wereobtained from the CLL Research Consortium (CRC) tissue bank (San Diego,Calif.). The blood samples were collected after obtaining informedconsent. PBMC were isolated by density gradient centrifugation usingHistopaque 1077 (Sigma, St. Louis, Mo. 63178). All patients met the NCIIWCLL criteria for diagnosis of CLL. The samples used contained ≧95%CD19 and CD5 positive cells, as assessed by flow cytometry. CLL sampleswere cultured in RPMI media containing 10% fetal bovine serum (FBS)(HyClone, Logan, Utah 84321 or Mediatech Inc., Herndon, Va. 20171) at37° C. in 5% CO2:95% air.

Gossypol and Apogossypol Preparation and Formulation

Apogossypol (NSC736630) was co-crystallized with ascorbic acid at 1:1molar ratio. Gossypol (NSC19048) was lyophilized in acetic acid form.Both compounds were provided by NCI-DTP (RAID-program). Compounds weredissolved in 100% sesame oil just before oral administration.Vehicle-control consisted of corresponding concentration of ascorbicacid suspended in 100% sesame oil.

Mouse Experiments

Gossypol and apogossypol were administered orally to mice daily at dosesof 0.06 mmol/kg or 0.12 mmol/kg, using a straight-type oral gavageneedle (18G-3″ Straight 2.25 mm ball, Braintree Scientific, Inc.). Thevolume of administration was 10 ml/kg, i.e., typically 0.2 mL per 20 gmmouse. Normal Balb/c mice of 7 to 8 weeks of age at the initiation ofthe study were employed for toxicity studies, while BCL-2 transgenicmice on Balb/c background of >6 months age were employed for efficacystudies. Age-matched, sex-matched mice were typically dosed 5 timesweekly, using a regiment of daily dosing 5 consecutive days (Mondaythrough Friday), followed by resting for 2 days, before resuming dosing.For BCL-2 transgenic mice, spleen-size was longitudinally monitoredeither by Ultrasound Imaging (Visualsonics) weekly and by physicalexamination using a digital caliper. At conclusion of treatments, micewere sacrificed via intra-peritoneal (i.p.) injection of 0.7 ml ofAvertin and whole blood was collected into Yellow-Top Serum Separatortubes (Becton Dickinson Vacutainer Systems Becton Dickinson and Company,Franklin Lakes, N.J. 07417-1885). Spleens were removed and weighed.

Hematology Studies

Whole blood (250 μl) was collected in EDTA-coated glass tubes (purpletop; MICROTAINER Brand Tube with EDTA, Catalogue #365973, Becton,Dickinson and Company, New Jersey 07417-1885) via either cardiacpuncture or severing the brachial artery of anesthetized mice. Afterthorough mixing, specimens were analyzed using a VetScan HM2 (AbaxisInc., Union City, Calif. 94587) hematology analyzer, measuring whiteblood cell count (WBC), red blood cell count (RBC), platelet (PLT)count, leukocyte differential (including % lymphocyte, % monocyte and %granulocyte), hematocrit (Ht), and hemoglobin (Hb).

FIGS. 6A-6C illustrate hematological profiles of mice treated withapogossypol or gossypol. Mice were orally administered with apogossypol,gossypol or vehicle-control at a daily dose of 0.12 mmol/kg, QDx5 forthree weeks (6 mice per group). Hematological profiles were analyzed bythe use of an automated HM2 hematology analyzer (Abaxis Inc., UnionCity, Calif. 94587) at conclusion of therapy with vehicle-control orapogossypol or at the time of death in mice treated with gossypol.

FIG. 6A shows WBC (left Panel) and RBC (right panel). As can be seen,both WBC and RBC were unaffected by treatments with gossypol andapogossypol. FIG. 6B shows data for hemoglobin (Hb)(left panel) and forhematocrit (Ht)(right panel). As can be seen, both Hb and Ht wereunaffected by treatments with gossypol and apogossypol. Finally, FIG. 2Cprovides data for lymphocyte count (left panel) and for platelet count:(right panel). As can be seen, gossypol induced lymphopenia, whereasapogossypol did not induce lymphopenia in Balb/c mice.

Serum Chemistry

Approximately 500 μl of whole blood was collected in glass tubes(yellow-top; MICROTAINER Brand, Serum Separator Tube, Catalogue #365956,Becton Dickinson and Company, Franklin Lakes, N.J. 07417-1885) and kepton ice for 30 minutes, then centrifuged at 12,000 r.p.m. (EppendorfCentrifuge 5415C) for 2 minutes to separate serum from cells and fibrinclot. The resulting serum specimens were analyzed using an automatedblood chemistry analyzer (“COBAS MIRA Classic”; Roche, Indianapolis,Ind. 46250-0414) to measure alanine aminotransferase (ALT) and aspartateaminotransferase AST), blood urea nitrogen (BUN), and Creatine.

FIG. 7 provides the experimental data illustrating reactive bloodchemistry profiles of mice treated with apogossypol or gossypol. Micewere orally administered with apogossypol, gossypol or vehicle-controlat a daily dose of 0.12 mmol/kg, QDx5 for three weeks (6 mice pergroup). As can be seen, gossypol induced elevation of serum levels ofALT and apogossypol was less hepato-toxic than gossypol.

Ultrasound Imaging

Stomach and intestines were examined also imaged by ultrasound forevidence of dilation, an indication of GI toxicity. Briefly, mice wereanesthetized using a mixture of isofluorane (5%) and oxygen gas (95%),restrained on a heated table using Aquagel Lubrication Gel (ParkerLaboratories, Inc., Fairfield, N.J. 07004), and abdominal hair wasremoved with a chemical depilation agent (Nair™ Hair Removal, Church &Dwight Co., Inc., Princeton, N.J. 08543). Aquasonic 100 UltrasoundTransmission Gel (Parker Laboratories, Inc., Fairfield, N.J. 07004) wasapplied to the abdomen prior to imaging using a high-frequency probe toassess gas and intestinal distention.

Cardiac Toxicity

Immediately after ultrasound imaging, electrocardiogram (ECG) analysisof anesthetized mice was performed using a MP150 Biopack System.

Histology

Vital organs, including liver, kidneys, spleen, heart, stomach, smallintestines, large intestines and lungs, were fixed in z-FIX solution for3 days, rinsed 3 times with phosphate-buffered saline (PBS) [pH 7.4],and then embedded in paraffin-blocks. Thin sections were cut (0.5 um),stained with hematoxylin-eosin (H&E), and evaluated by light microscopyfor histological abnormalities. In addition, unstained sections wereanalyzed by the terminal deoxynucleotidyl transferase end-labeling(TUNEL) method to stain cells with DNA fragmentation indicative ofapoptosis.

Splenocyte Isolation

Spleens were excised from sacrificed mice and cell suspensions treatedwith a mouse erythrocyte lysing kit (R & D Systems). Total splenocytecount was determined by trypan blue dye exclusion assays usinghemocytometers. The percentage of B-lymphocytes was determined byfluorescence activated cell sorter (FACS) analysis (FACS-CANTO,Bectin-Dickinson Inc., Mountain View, Calif.) following staining cellswith Phyco-Erythrin (PE)-conjugated anti-CD19 or -B220 antibodies(Becton Dickinson, San Jose, Calif. 95131).

Cell Culture and Cytotoxicity Studies

Splenocytes were suspended at 1×10⁶ cells/mL in RPMI 1640 medium(Mediatech Inc., Herndon, Va. 20171) containing 10% fetal bovine serum(Mediatech Inc., Herndon, Va. 20171) and Penicillin/Streptomycin(Mediatech Inc., Herndon, Va. 20171). Human B-CLL cells and 3 B-NHL celllines, including RS11846, DOHH2 and 380 cells, were cultured in RPMI1640 medium (Mediatech Inc., Herndon, Va. 20171) containing 10% fetalbovine serum (Mediatech Inc., Herndon, Va. 20171) andPenicillin/Streptomycin (Mediatech Inc., Herndon, Va. 20171). Cells werecultured with various concentrations of Gossypol, ApoGossypol, orascorbic acid for 1-2 days. The percentage of viable cells wasdetermined by Annexin V- and propidium iodide (PI)-labeling, using anApoptosis Detection kit (BioVision Inc.), analyzing stained cells byflow cytometry (FACSort; Bectin-Dickinson, Inc.; Mountain View, Calif.).Cells that were annexin-V-negative and PI-negative were consideredviable.

FIG. 8 provides the experimental data illustrating a comparison ofapoptosis induction of NHL B-cell lines, including DOHH2, RS11846 and380, by apogossypol and gossypol. NHL B-cell lines, including DOHH2,RS11846 and 380, were cultured in RPMI medium containing 10% fetalbovine serum (FBS) for 48 hours, in the absence and presence of variousconcentrations of gossypol and apogossypol as indicated in the figures.

After 48 hours of incubation, % viability was determined by FACSortafter staining cells by the use of an Annexin V-FITC/PI ApoptosisDetection kit (BioVision Inc.). Viable cells were defined by AnnexinV-negative, PI-negative cells. DOHH2 and RS11846 cell lines wereslightly more sensitive to gossypol and apogossypol in vitro with IC50of approximately 3 μM, whereas 380 cell line was slightly more resistantto gossypol and apogossypol. In all three NHL B-lymphoma cell lines,apogossypol was slightly more potent than gossypol, but their potencieswere roughly comparable.

FIG. 9 provides the experimental data illustrating a comparison ofactivity of gossypol and apogossypol against cultured murine B-cellsfrom transgenic mice: BCL-2 vs. BCL-2/TRAF2DN. Spleen tissues wereremoved from BCL-2 transgenic mice and BCL-2/TRAF2DN mice, andsplenocytes were isolated by the use of a mouse erythrocyte lysing kit(R & D Systems) according to the manufacturer's manual.

Splenocytes were cultured in RPMI medium containing 10% fetal bovineserum (FBS) for 18 hours, in the absence and presence of variousconcentrations of gossypol and apogossypol as indicated in the figures.After 18 hours of incubation, % viability of splenocytes was determinedby FACSort after staining cells by the use of an Annexin V-FITC/PIApoptosis Detection kit (BioVision Inc.). Viable cells were defined byAnnexin V-negative, PI-negative cells. In BCL-2 transgenic mouse;apogossypol was several-fold more potent than gossypol in induction ofapoptosis against cultured B-cells with IC50 of roughly 1-2 μM forapogossypol vs. 10 μM for gossypol. In contrast, murine B-cells fromBcl-2/TRAF2DN mice were roughly 10-fold more resistant to bothapogossypol and gossypol than Bcl-2 transgenic mice.

FIG. 10 provides the experimental data illustrating a comparison ofapogossypol and gossypol induction of apoptosis of cultured CLL B-cells.CLL samples were incubated in RPMI media containing 10% fetal bovineserum (FBS) at 37° C. with 5% CO2 for 48 hours, in the absence orpresence of various concentrations of gossypol and apogossypol asindicated in the figures.

After 48 hours of culture, % viability was determined by FACSort afterstaining cells by the use of an Annexin V-FITC/PI Apoptosis Detectionkit (BioVision Inc.). Viable cells were defined by Annexin V-negative,PI-negative cells. Apogossypol was approximately 3-fold more potent thangossypol against cultured CLL B-cells in vitro. There was significantdifference in apoptosis induction between apogossypol group and gossypolgroup (p<0.025) by two-way ANOVA analysis.

FIGS. 11A and 11B provide the experimental data illustrating apogossypolactivity in Bcl-2 transgenic mice. FIG. 11A shows the results of a lowdose study (at 0.06 mmol/kg) and FIG. 11B—a high dose study (at 0.12mmol/kg). Age-matched and sex-matched BCL-2 transgenic mice were usedfor efficacy studies. BCL-2 transgenic mice spontaneously developed lowgrade B-cell lymphoma as characterized by splenomegaly, as a function oftime. In BCL-2 transgenic mice, the disease progression can be dividedinto two stages; at the first stage, the disease is characterized bysplenomegaly as a result of expansion of B-cells in spleen due toover-expressed BCL-2 in B6 mice, and at the second stage, anothergenetic hit(s) may strike, resulting in disseminated lymphoma ascharacterized by bulky lymphadenopathy as well as splenomegaly.

In this study, BCL-2 transgenic mice at the first stage were used forthis efficacy study. In a separate study with untreated BCL-2 transgenicmice, wet weight of spleen ranged from 195-mg to 335-mg, and wet weightof spleen was found to be nearly comparable in age-matched, sex-matchedBCL-2 transgenic mice. Apogossypol stabilized with ascorbic acid at 1:1molar ratio, gossypol stabilized with acetic acid at 1:1 molar ratio andvehicle-control (ascorbic acid in 100% sesame oil) were orallyadministered to BCL-2 transgenic mice once daily (QDX5) for 3 weeks,consecutively. At conclusion of treatment, BCL-2 transgenic mice weresacrificed via intraperitoneal injection of 0.7 ml of avertin(anesthetic solution) and spleen was removed and weighed. Splenocyteswere isolated by the use of mouse erythrocyte lysing kit (RD Systems).Total splenocyte count was determined by trypane blue exclusion assays.% B-cell count was determined by FACS analysis after staining cells withCD-5, a B-cell marker. Reactive data are shown by FIGS. 11A and 11B.

As can be seen from FIG. 11A, at a low dose of 0.06 mmol/kg, bothgossypol and apogossypol were well tolerated and induced shrinkage ofsplenomegaly, as evidenced by reductions in wet weight of spleen as wellas B-cell count in spleen. Apogossypol induced shrinkage of spleen to asignificant extent (p<0.03 for wet weight of spleen, P<0.05 for splenicB-cell counts), whereas gossypol induced shrinkage of spleen to aconsiderable extent but not significantly.

As can be seen from FIG. 11B, at a high dose of 0.12 mmol/kg, gossypolwas not tolerated in BCL-2 transgenic mice, whereas apogossypol was welltolerated in Bcl-2 transgenic mice at a high dose of 0.12 mmol/kg.Apogossypol induced shrinkage of splenomegaly to a significant extent(p<0.001 for wet weight of spleen, P<0.0001 for splenic B-cell counts).Age-matched, sex-matched BCL-2 transgenic mice were evaluated forshrinkage of spleen after conclusion of apogossypol therapy and spleensize was reduced roughly by half at a daily dose of 0.12 mmol/kg.

Example 19 Evaluation of the Cytotoxic Activity of the Compounds onHuman Tumors Cells

This example illustrates the efficacy of gossypol on human tumor cells.To evaluate the cytotoxic activity of the compounds on human tumorscells, their biological activities were tested using XTT dye reductionassays using two breast cancer cell lines: MCF7 (high expressor ofBCL-2/BCL-X_(L)) and ZR75-1 (low expressor of BCL-2/BCL-X_(L)). Gossypolis a cytotoxic agent for MCF7 and ZR75-1 cells, reducing cell viabilityin a dose-dependent manner, with IC₅₀ values of 13.2 μM and 8.4 μMrespectively. Purpurogallin, however, did not show appreciable activityin these assays, potentially due to its hydrophilic character (ClogP−0.7).

Consistent with this observation, a purpurogallin derivative 5D1 that ispredicted to have better cell-membrane permeability properties (based onits ClogP of ˜2.5) reduced cell viability in a dose-dependent manner,with IC₅₀ value of ˜50 μM the ZR75-1 cell line (not shown). Therefore,the cellular activities of the compounds were evaluated in HeLa cells,which are known to be less selective for compounds uptake. Theinhibition data obtained with HeLa cells viability assays parallel thein vitro binding data with BCL-X_(L), with a correlation coefficient ofr=0.9 (p=0.001).

Docking studies with FlexX software (Kramer et al., Proteins, 37:228(1999)) implemented in Sybyl (TRIPOS) using the BCL-X_(L) conformationfound in the complex with Bak-peptide showed an optimal location forgossypol in the deep hydrophobic cleft normally occupied by the Bakhelical BH3 peptide in the complex. Both the (+) and the (−)stereoisomers of gossypol were docked, as these exhibited differentactivity in previous cell-based assays which showed that (−) gossypol isten times more effective than (+) gossypol as a cytotoxic agent. Thegoodness of the fit as measured by a scoring function, and theintermolecular energy after minimization with the DOCK routine of Sybyl,was considerably better for (−) gossypol (−32.7 Kcal/mol) versus (+)gossypol (−25 Kcal/mol), in agreement with these observations. Theoverall positioning of both stereoisomers of Gossypol is very similar.

Fluorescence Polarization Assays (FP A)

FP A assays were conducted with a fluorescein-labeled Bad peptide (NL WAAQRYGRELRRMSD-K(FITC)-FVD) (Synpep Corporation, Dublin, Calif.) using aLJL Analyst HT (Molecular Devices Co., Sunnyvale, Calif.). Dilutionbuffer for all stocks and samples was 50 μM Tris-Bis pH 7.4, 0.01%bovine gamma globulin. A series of two-fold dilutions of Gossypol wereprepared, i.e., 100 μM, 50 μM, down to 0.1 μM in dilution buffer. Toeach tube was added a solution containing 30 nM of BCL-X_(L) and 4 nMfluoresceinated peptide. The tubes were incubated for 5 minutes at roomtemperature and 20 μl each of reaction mixture was transferred to96-well black PS, HE Microplate (LJL Biosystems Co). All assays wereperformed in quadruplicate, with blank wells receiving no Gossypol.Then, the plate was read for total intensity and polarization (in mPunits) was measured. Controls included dose-responses measurements inabsence of the proteins, to assess any interactions between thecompounds and the FITC-BH3 peptide. Eventual effects were taken intoaccount by subtraction.

NMR Spectroscopy

2D [¹⁵N, ¹H]-TROSY spectra for BCL-X_(L) were measured with 0.5 mMsamples of ¹⁵N-labeled BCL-X_(L). ¹⁵N-labeled and unlabeled BCL-X_(L)were prepared and purified according to known methods. Forchemical-shift mapping and docking studies the three-dimensionalstructure of BCL-X_(L) in complex with Bak peptide (PDB code 1BXL) wasused. In addition to chemical-shift mapping with labeled proteins,T_(1p) measurements and saturation transfer experiments such asWaterLOGSY experiments were also performed to further validate thebinding of the studied compounds to BCL-X_(L).

All experiments were performed with a 500 MHz Varian Unity+ spectrometeror a 600 MHz Bruker Avance600 spectrometer, both equipped with four rfchannels and z-axis pulse-field gradients. Selective water saturationwas performed with a train of selective IBURP2 pulses of 7 ms durationsspaced by a 10 ms delay. Total saturation time used was 2.5s T_(1p)series were measured with a spin-lock pulse of variable length.Measurements were then performed with 1 ms, 10 ms, 50 ms, 150 ms, 200ms, 250 ms and 300 ms spin-lock time with 100 μM compounds in theabsence and presence of 10 μM protein. In all experiments, de-phasing ofresidual water signals was obtained with a WATERGATE sequence.

Molecular Modeling

Molecular modeling studies were conducted on several R12000SGI Octaneworkstations with the software package Sybyl version 6.9 (TRIPOS). Thedocked structure of Gossypol was initially obtained by FlexX asimplemented in Sybyl. Two calculations were performed. In the first, allbinding-site torsion angles were kept fixed, while in the secondside-chain torsion angles were free to change. The average scoringfunction for the 30 best solutions was slightly lower when theside-chains were free to rotate. The position of the side-chains in themodel did not change substantially from the initial values. The scoringfunction for (+) gossypol was inferior to (−) gossypol, but the overallpositioning of both stereoisomers was very similar.

The resulting best scoring structures were subsequently energy minimizedby using the routine DOCK of SYBYL keeping the site rigid. The energy ofthe ligands after the DOCK minimization was within 5 Kcal/mol from theirglobal minimum of energy. Superposition of compounds was obtained by theroutine MULTIFIT of SYBYL. Color figures showing three-dimensionalstructures were prepped with the programs SYBYL and MOLMOL.

Inhibitory Effect of Compounds on Cancer Cell Survival

The effects of the compounds on viability of tumor cells in culture weremonitored by using XTT assays with MCF7 and ZR75-1 cell lines. MCF7cells were grown in DMEM containing 10% fetal bovine serum,penicillin/streptomycin, supplemented with 10⁻¹⁰ M insulin, 1 mM sodiumpyruvate and glutamine. ZR75-1 cells were grown n RPMI containing 10%fetal bovine serum, penicillin/streptomycin, supplemented with HEPESbuffer, 1 mM sodium pyruvate and glutamine. Cells were regularly testedfor mycoplasma contamination. Cells were seeded triplicates at aninitial cell density of 1,000 cells per well. Blank wells received nocells.

Gossypol, purpurogallin and 5D1 were added at final concentrations of 0,1, 10 and 100 μM and incubated for three days. Relative numbers ofviable cells was determined by XTT assay. Briefly, in a 96-well plate,we added 50 μl of a mixture of 1 mg/ml of XTT(2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazoliumhydroxide) (Polysciences, Washington, Pa.) containing 0.025 mM PMS(phenazine methosulfate) to each well. The 96-well plates werereincubated for an additional 4 hours to allow for XTT formazanproduction. Then, the contents of each plate were mixed and opticaldensities were determined at a wavelength of 450 nm (OD₄₅₀). Net OD₄₅₀was determined after subtracting OD₄₅₀ of blank wells. Low-passage HeLacells (between passage number 10 and 20) were transfected with pcDNA3-BCL-X_(L) or control pcDNA3 plasmids using Lipofectamine Plus reagent(Invitrogen) and selected in medium containing 800 μg/ml of G418.Immunoblot analysis of BCL-X_(L) was accomplished as previouslydescribed. HeLa-transfectants were treated with various doses ofgossypol, purpurogallin, and its derivatives (0, 1, 3, 10 and 100 μM).

Chemicals

Pure polyphenols were obtained from SIGMA (gossypol and purpurogallin)and/or from Microsource Discovery Systems (Purpurogallin derivatives).Reference compounds were obtained from Chembridge Corp. (San Diego).Gossypol was tested as a racemic mixture of (+) and (−) isomers.Compounds were dissolved in DMSO at 100 mM concentration and stored at−20 DC. NMR analysis was periodically performed on the compounds as aquality control, prior to further dilution for binding and displacementassays. Reactivity of Gossypol was tested with a 15N-labeled testprotein (BIR3 domain of XIAP). A solution containing 1 mM gossypol and200 μM N-labeled BIR3 was incubated for two hours and the[¹⁵N,¹H]-correlation spectrum was recorded and compared with thespectrum of the apo-Bir3. No appreciable differences in the spectra wereobserved. Results are summarized in Table 12.

TABLE 12 STRUCTURE ACTIVITY RELATIONSHIPS (SAR) OF PURPUROGALLINDERIVATIVES IC₅₀ (μM) IC₅₀ (μM) CMPD R₁ R₂ R₃ R₄ R₅ (BCL-X_(L)) (HeLa)Purpurogallin —OH —OH —OH —OH —H 2.2 6.5 5D1 —H —OH —OH —OH —COOC₂H₅ 7351.5 1163 —H —OH —OH —OH —COOCH₃ 2.6 ~30 1142 —H —OH —OH —OH —COOH 7.422.9 6A1 —OCH₃ —OCH₃ —OCH₃ —OCH₃ —H >100 >100 6A7 —OCH₃ —OCH₃ —OCH₃—OCH₃ —H >100 >100

Example 20 Maximum Tolerated Dose (MTD)

Young female Balb/c mice (7-weeks-old) were injected with 100 mg/kg, 75mg/kg, 50 mg/kg, 25 mg/kg, 12.5 mg/kg and 6.25 mg/kg of compound 8rintraperitoneally (one mouse per dose) and observed for survival, vitalsigns, weight loss, etc. for 14 days, in compliance with MTD generalprotocol proposed by DTP (Developmental Therapeutics Program) at NCI.Compound 8r was first dissolved in 100% ethanol, supplemented byCremophore EL and saline, just before injection, with a ratio ofEthanol:Cremophore EL:Saline=10:10:80. Upon conclusion of the study,mice were euthanized by CO2, and vital organs were harvested and fixedwith z-FIX solution for 3 days at room temperature, rinsed in PBS threetimes, for further histological evaluation.

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Although the disclosure has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the disclosure. Accordingly,the disclosure is limited only by the following claims.

1. A compound having structure A, or a pharmaceutically acceptable saltthereof:

wherein: each R is independently C(O)NHX; and X is (C₁-C₆)alkyl,substituted (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, substituted(C₃-C₈)cycloalkyl, aryl, substituted aryl, (C₁-C₆)alkylaryl, substituted(C₁-C₆)alkylaryl, wherein each X substituent is independentlysubstituted or unsubstituted (C₁-C₆)alkyl, trifluoromethyl, halogen,substituted or unsubstituted phenyl or phenoxy.
 2. The compound of claim1 or a pharmaceutically acceptable salt thereof, wherein X is(C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, aryl, or (C₁-C₆)alkylaryl.
 3. Thecompound of claim 1 or a pharmaceutically acceptable salt thereof,wherein X is substituted (C₁-C₆)alkyl, substituted (C₃-C₈)cycloalkyl,phenyl, substituted phenyl, or substituted (C₁-C₆)alkylaryl.
 4. Thecompound of claim 1 or a pharmaceutically acceptable salt thereof,wherein each R is independently


5. The compound of claim 1 or a pharmaceutically acceptable saltthereof, wherein each R is independently C(O)NHCH₂CH(CH₃)C₆H₅.
 6. Thecompound of claim 1 or a pharmaceutically acceptable salt thereof,wherein X is (C₁-C₆)alkylaryl or substituted (C₁-C₆)alkylaryl.
 7. Thecompound of claim 6 or a pharmaceutically acceptable salt thereof,wherein X is substituted or unsubstituted benzyl.
 8. The compound ofclaim 1 or a pharmaceutically acceptable salt thereof, wherein thecompound or pharmaceutically acceptable salt is:


9. A method for treating breast cancer, colon cancer, lung cancer,ovarian cancer, prostate cancer, renal cancer, leukemia, lymphoma, ormelanoma, the method comprising the step of administering to a subjectin need thereof a therapeutically effective amount of a compound havingstructure A of claim 1, or a pharmaceutically acceptable salt thereof,thereby treating the cancer.
 10. The method of claim 9, wherein thecancer is lung cancer, breast cancer, prostate cancer, or lymphoma. 11.The method of claim 9, wherein the treatment includes inhibition ofactivity of at least one BCL-2 family protein.
 12. The method of claim9, further comprising administering the compound having structure A incombination with an anti-cancer agent.
 13. A method of treating breastcancer, colon cancer, lung cancer, ovarian cancer, prostate cancer,renal cancer, leukemia, lymphoma, melanoma or an autoimmune disease in asubject having at least one elevated BCL-2 family protein expressionlevel, the method comprising the step of administering to the subject atherapeutically effective amount of a compound having structure A ofclaim 1, or a pharmaceutically acceptable salt thereof.
 14. The methodof claim 13, further comprising determining whether the subject isresponsive to the treatment that utilizes the compound having structureA, comprising determining the level of at least one of the BCL-2 familyprotein in the subject and comparing to a normal control sample, whereinan elevated level is indicative of a subject responsive to the treatmentthat utilizes the compound having structure A, or a pharmaceuticallyacceptable salt thereof.
 15. The method of claim 14, wherein thedetermination is made based on a sample from the subject.
 16. A methodof determining whether a subject is responsive to a therapy for treatingbreast cancer, colon cancer, lung cancer, ovarian cancer, prostatecancer, renal cancer, leukemia, lymphoma, or melanoma that utilizes thecompound having structure A of claim 1, or a pharmaceutically acceptablesalt thereof, the method comprising the step of determining the level ofat least one of the BCL-2 family protein in the subject and comparing toa normal control sample, wherein an elevated level is indicative of asubject responsive to the therapy that utilizes the compound havingstructure A, or a pharmaceutically acceptable salt thereof.
 17. Themethod of claim 16, wherein the determination is made based on a samplefrom the subject.
 18. The method of claim 16, wherein the sample is abiological fluid or tumor sample.
 19. The method of claim 16, whereinthe BCL-2 family polynucleotide or polypeptide is selected from BCL-2,BCL-XL, BCL-W, MCL-1, and BCL-A1.
 20. A method of inducing apoptosis ina cancer or an immune cell having a level of at least one of the BCL-2family protein member greater than levels in a control cell, wherein thecancer cell is a breast cancer cell, colon cancer cell, lung cancercell, ovarian cancer cell, prostate cancer cell, renal cancer cell,leukemia cell, lymphoma cell, or melanoma cell, the method comprisingthe step of administering to the cell an effective amount of a compoundhaving structure A of claim 1, or a pharmaceutically acceptable saltthereof, to reduce the level of BCL-2 family protein(s) and induceapoptosis in the cell.
 21. The method of claim 20, wherein the cell is acell of the immune system.
 22. A method of determining the effectivenessof a therapeutic regimen for treating breast cancer, colon cancer, lungcancer, ovarian cancer, prostate cancer, renal cancer, leukemia,lymphoma, melanoma or an autoimmune disease, including administration ofa compound having structure A of claim 1, or a pharmaceuticallyacceptable salt thereof, in a subject, the method comprising the step ofcomparing the level of a BCL-2 family protein in a cell of the subjectprior to and during treatment with the compound having structure A, or apharmaceutically acceptable salt thereof, wherein a decreased level ofBCL-2 family protein is indicative of effectiveness of the therapy thatutilizes the compound having structure A, or a pharmaceuticallyacceptable salt thereof.
 23. A method of treating inflammation in asubject, the method comprising the step of administering to the subjectin need of the treatment a pharmaceutically effective amount of acompound having structure A of claim 1, or a pharmaceutically acceptablesalt thereof, wherein the subject is afflicted with lupus erythmatosus,psoriasis, psoriatic arthritis, lupus nephritis, rheumatoid arthritis,multiple sclerosis, ulcerative colitis, myasthenia gravis, ITP, TTP,Grave's disease, Hashimoto's thyroiditis, Crohn's disease, autoimmunehemolytic anemias, insulin dependent diabetes mellitus,glomerulonephritis, rheumatic fever, osteoarthritis, gouty arthritis,dermatitis, bronchitis, rhinitis, asthma, Sjogren' syndrome, meningitis,adrenoleukodystrophy, CNS vasculitis, mitochondrial myopathies,Amyotrophic Lateral Sclerosis, Alzheimer's disease, or a tumor, toreduce the inflammation thereby.
 24. The method of claim 23, wherein themitochondrial myopathy is MELAS syndrome, MERF syndrome, Leber'sdisease, Wernicke's encephalopathy, Rett syndrome, homocystinuria,hyperprolinemia, nonketotic hyperglycinemia, hydroxybutyricaminoaciduria, sulfite oxidase deficiency, or combined systems disease(B12 deficiency).
 25. The method of claim 23, further comprisingadministering a selective serotonin reuptake inhibitor (SSRI).